Mice that make heavy chain antibodies

ABSTRACT

Genetically modified non-human animals and methods and compositions for making and using them are provided, wherein the genetic modification comprises a deletion in an immunoglobulin constant region CH1 gene (optionally a deletion in a hinge region) of an IgG, IgA, IgD, and/or IgE, and wherein the mouse is capable of expressing a functional IgM. Genetically modified mice are described, including mice having a functional IgM gene and modified to have a deletion of a CH1 domain and a hinge region in a heavy chain constant domain that is not an IgM, e.g., in an IgG heavy chain constant domain. Genetically modified mice that make human variable/mouse constant chimeric heavy chain antibodies (antibodies that lack a light chain), fully mouse heavy chain antibodies, or fully human heavy chain antibodies are provided.

This application is a continuation of U.S. patent application Ser. No.14/267,279, filed May 1, 2014, now U.S. Pat. No. 9,888,675, which is adivisional of U.S. patent application Ser. No. 12/965,050, filed 10 Dec.2010, now U.S. Pat. No. 8,754,287, which claims the benefit of thefiling date under 35 USC § 119(e), and is a nonprovisional, of U.S.Provisional Patent Application Ser. No. 61/285,250, filed 10 Dec. 2009,each of which applications is hereby incorporated by reference.

FIELD OF INVENTION

The field of invention is genetically modified non-human animals thatmake heavy chain antibodies, in particular genetically modified animalsthat comprise a nucleotide sequence deletion in a sequence encoding aCH1 domain (or CH1 domain and hinge region) of an immunoglobulin gamma(IgG) gene but that are capable of expressing an IgM that does not lacka functional CH1 domain, and in particular mice that are capable ofmaking wild-type IgM molecules (i.e., with CH1 domains) but that makeheavy chain IgG antibodies devoid of a functional CH1 domain (or CH1domain and hinge region).

BACKGROUND

In most animals, normal immunoglobulin heavy chains are onlywell-expressed when coupled with their cognate light chains. In humans,lone heavy chains are found in heavy chain disease that is manifested bydysfunctional heavy chains that lack sequences of the variable heavy,the CH1, or the variable heavy and CH1 domains. Heavy chains devoid oflight chains are encountered in certain species of fish and in camels.Such heavy chains lack a functional CH1 domain and have non-humanfeatures in their heavy chain variable domains. Attempts have been madeto make camelized antibodies by modifying mice to express camelizedgenes that mimic VHH domains found in camels or certain species of fish,in part by removal of IgM and IgG CH1 domains and conforming the heavychain variable regions to resemble those of camels and/or certainspecies of fish. However, camelized antibodies would be expected toinduce immune responses in non-camel animals.

There is a need in the art for genetically modified non-human animalsthat make heavy chain antibodies that have non-camelid VH domains.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a wild-type IgG1 locus in a mouse (IgG1, top),showing the JH region gene segment fusing to a CH1 gene segment,followed by a hinge region, a CH2 gene segment, and a CH3 gene segment;an IgG1 locus targeted with a construct that deletes a CH1 domain(IgG1ΔCH1, middle); and an IgG1 locus targeted with a construct thatdeletes both a CH1 domain and a hinge region (IgG1ΔCH1-Δhinge, bottom).

FIG. 2 illustrates targeting a mouse IgG1 gene to make a geneticallymodified locus that expresses an IgG1 lacking a CH1 domain.

FIG. 3 illustrates targeting a mouse IgG1 gene to make a geneticallymodified locus that expresses an IgG1 lacking a CH1 domain and lacking ahinge region.

FIG. 4 illustrates targeting a mouse heavy chain constant region locusto make a genetically modified locus that expresses an IgG1 lacking aCH1 domain, and does not express an IgG2b or an IgG2a.

FIG. 5 illustrates a mouse heavy chain constant region targeted with aconstruct that deletes a CH1 domain and deletes a hinge region and thatdeletes an IgG2b gene and an IgG2a gene.

FIG. 6 illustrates a heavy chain constant region of a geneticallymodified mouse having an IgG1 that lacks a CH1 domain or lacks a CH1domain and a hinge region (top), and a heavy chain constant region of agenetically modified mouse having an IgG1 that lacks a CH1 domain orlacks a CH1 domain and a hinge region, and that lacks an IgG2a gene andlacks an IgG2b gene (bottom).

FIG. 7 shows Western blots of CHO cell supernatants from CHO cellsengineered to independently express control (cytokine ectodomain fusionwith a mouse Fc), chimeric (human VR)/(mouse Fc) heavy chain antibodylacking a CH1 domain (hVR-mFcΔCH1), camelized chimeric (human VR)/(mouseFc) heavy chain antibody lacking a CH1 domain (hVR*-mFcΔCH1), chimeric(human VR)/(mouse Fc) heavy chain antibody (hVR-mFc), camelized chimeric(human VR)/(mouse Fc) heavy chain antibody (hVR*-mFc), mFc with (mFc) orwithout (mFcΔCH1) a CH1 domain.

FIG. 8 shows Western blot images from a reducing SDS-PAGE of mouse serafrom a wild-type mouse (left) and from a genetically modified mousewhose IgG1 lacks a CH1 domain and lacks a hinge region (heterozygous)(right), blotted with anti-mouse IgG; schematics of the heavy chains areprovided, as are molecular weight marker positions.

FIG. 9 shows Western blots images from a non-reducing SDS-PAGE of mousesera from a wild-type mouse (WT) and four genetically modified micewhose IgG1 lacks a CH1 domain and lacks a hinge region (homozygous;noted as HO 1, HO 2, HO 3, HO 4, respectively), blotted with anti-mouseIgG; each mouse (WT or HO) is represented by two lanes indicated bybrackets above the lanes corresponding to 1:5 and 1:10 dilutions ofserum for each animal (consecutive lanes from left to right for each).

FIG. 10 provides a schematic diagram of a normal IgG1 antibody (left)and a heavy chain antibody that lacks a CH1 domain and lacks a hingeregion.

FIG. 11 shows separate IgG1 and IgG2b serum immunoglobulin assays fromwild type mice (WT) and genetically modified mice that contain an IgG1lacking a CH1 domain and lacking a hinge region (HO; homozygous mousethat expresses a heavy chain antibody that lacks a CH1 domain and lacksa hinge region). Control is pooled human serum.

FIG. 12 shows the protein sequences of eleven independent RT-PCR clonesamplified from splenoctye RNA of mice bearing mouse heavy chain genesequences at a modified endogenous mouse heavy chain locus devoid ofIgG1 CH1 and hinge region sequences. B1=SEQ ID NO:19; B2=SEQ ID NO:21;B3=SEQ ID NO:23; B5=SEQ ID NO:25; D2=SEQ ID NO:27; D5=SEQ ID NO:29;D6=SEQ ID NO:31; E2=SEQ ID NO:33; E8=SEQ ID NO:35; E10=SEQ ID NO:37;F6=SEQ ID NO:39. Lower case bases indicate non-germline bases resultingfrom either mutation and/or N addition during recombination. Dotsrepresent artificial gaps in the sequence for proper alignment offramework (FR) and complementary determining regions (CDR), which arenoted above the sequences. The first nine amino acids from the CH2region of the endogenous IgG1 (CH2) constant region are shown for eachclone.

FIG. 13 shows the protein sequences of seven independent RT-PCR clonesamplified from splenoctye RNA of mice bearing human heavy chain genesequences at a modified endogenous mouse heavy chain locus devoid of anIgG1 CH1 region sequence. A8=SEQ ID NO:51; C2=SEQ ID NO:53; D9=SEQ IDNO:55; C4=SEQ ID NO:57; H8=SEQ ID NO:59; A5=SEQ ID NO:61; A2=SEQ IDNO:63. Lower case bases indicate non-germline bases resulting fromeither mutation and/or N addition during recombination. Dots representartificial gaps in the sequence for proper alignment of framework (FR)and complementary determining regions (CDR), which are noted above thesequences. The first seven amino acids of the 13 amino acid hinge regionof the endogenous IgG1 (HINGE) constant region are shown for each clone.

SUMMARY

Genetically modified cells, non-human embryos, non-human animals andmethods and compositions for making and using them are provided, whereinthe animals are genetically modified to lack a functional CH1 sequencein an immunoglobulin G (IgG), optionally modified to lack a functionalIgG hinge region on the modified IgG, and wherein the cells, embryos,and animals comprise a functional IgM CH1 sequence. In some aspects, themice comprise a replacement of one or more, or all, endogenous mouseimmunoglobulin heavy chain variable region gene segments with one ormore human immunoglobulin heavy chain variable region gene segments. Insome aspects, all endogenous mouse V, D, and J gene segments arereplaced with one or more human V, one or more human D, and one or morehuman J gene segments.

In one aspect, a genetically modified mouse is provided, wherein thegenetic modification comprises a modification of a nucleotide sequenceencoding an IgG constant region, wherein the modification results in aloss of function of the CH1 domain of the IgG constant region. In oneembodiment, the loss of function modification is a deletion of anucleotide sequence encoding the CH1 domain, or a deletion within thenucleotide sequence encoding the CH1 domain.

In one embodiment, the IgG is selected from IgG1, IgG2a, IgG2b, and acombination thereof. In one embodiment, the IgG is an IgG1. In oneembodiment, the IgG is an IgG1, an IgG2a, and an IgG2b.

In one embodiment, the modification further comprises a deletion of anucleotide sequence for a hinge region of the IgG that comprises the CH1modification.

In one embodiment, the genetically modified mouse is selected from a 129strain, a C57BL/6 strain, and a mix of 129×C57BL/6. In a specificembodiment, the mouse is 50% 129 and 50% C57BL/6.

In one embodiment, the genetically modified mouse is a 129 strainselected from the group consisting of a 129P1, 129P2, 129P3, 129X1,129S1 (e.g., 129S1/SV, 129S1/SvIm), 129S2, 129S4, 129S5, 129S9/SvEvH,129S6 (129/SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g., Festing etal. (1999) Revised nomenclature for strain 129 mice, Mammalian Genome10:836). In one embodiment the genetically modified mouse is a C57BLstrain, in a specific embodiment selected from C57BL/A, C57BL/An,C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ,C57BL/10, C57BL/10ScSn, C57BL/10Cr, C57BL/OIa. In a specific embodiment,the genetically modified mouse is a mix of an aforementioned 129 strainand an aforementioned C57BL/6 strain. In another specific embodiment,the mouse is a mix of aforementioned 129 strains, or a mix ofaforementioned BL/6 strains. In a specific embodiment, the 129 strain ofthe mix is a 129S6 (129/SvEvTac) strain.

In one embodiment, the mouse comprises one or more unrearrangedendogenous mouse heavy chain immunoglobulin variable region (mVR) genesegments operably linked to the modified IgG constant region sequence.In one embodiment, the one or more mVR gene segments are from a mouse VHgene family selected from VH1, VH3, VH5, VH7, VH14, and a combinationthereof. In one embodiment, the one or more mVR gene segments areselected from a mVH 1-26, 1-42, 1-50, 1-58, 1-72, 3-6, 5-6, 7-1, 14-2,and a combination thereof.

In one embodiment, the mouse comprises a rearranged gene that encodes anFR1, FR2, and an FR3 in an IgG heavy chain that lacks a functional CH1region, wherein the FR1, FR2, and FR3 are each independently at least90%, 95%, 96%, 97%, 98%, or 99% identical to an FR1, FR2, and FR3derived from a mVH germline sequence selected from a VH1, VH3, VH5, VH7,and VH14 gene family. In one embodiment, the mVH germline sequence isselected from a 1-26, 1-42, 1-50, 1-58, 1-72, 3-6, 5-6, 7-1, and 14-2sequence.

In one embodiment, the mouse comprises a CDR3 derived from a DH genesegment selected from DH 1-1, 2-14, 3-1, 3-2, 3-3, 4-1, and acombination thereof. In one embodiment, the mouse CDR3 comprises asequence encoded by a JH that is a JH1, JH2, JH3, or JH4.

In one embodiment, the mouse comprises a rearranged antibody sequencethat encodes a CDR3 that is derived from a rearrangement of a DH 1-1,2-14, 3-1, 3-2, 3-3, 4-1, and a JH1, JH2, JH3, or JH4.

In one embodiment, the mouse comprises a rearranged gene that encodes anFR4 in an IgG heavy chain that lacks a functional CH1 region, whereinthe FR4 is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to an FR4encoded by a rearrangement of a DH1-1, 2-14, 3-1, 3-2, 3-3, or 4-1 witha JH1, JH2, JH3, or JH4.

In one embodiment, the mouse comprises an unrearranged human heavy chainimmunoglobulin variable region (hVR) gene segment at an endogenous mouseheavy chain variable region locus. In one embodiment, the mousecomprises an unrearranged hVR gene segment operably linked to themodified IgG constant region sequence at an endogenous mouse heavy chainvariable region locus. In one embodiment, the hVR gene segments are froma human VH gene family selected from VH1, VH3, VH4, and a combinationthereof. In one embodiment, the one or more hVR gene segments areselected from 1-2, 1-8, 1-18, 1-46, 1-69, 3-21, 3-72, and 4-59. In aspecific embodiment, the one or more hVR gene segments are selected from1-8, 1-18, and 1-69.

In one embodiment, all or substantially all mouse heavy chain V genesegments are replaced by one or more human heavy chain V gene segments.In one embodiment, all mouse heavy chain V and D gene segments arereplaced by one or more human heavy chain V and D gene segments. In oneembodiment, all mouse heavy chain V, D, and J gene segments are replacedwith one or more human heavy chain V, one or more human heavy chain D,and one or more human heavy chain J gene segments. In these embodiments,the human heavy chain V and/or D and/or J gene segments are at the mouseendogenous heavy chain locus and are operably linked to the mouseconstant region gene(s) or modified mouse constant region gene(s).

In one embodiment, the mouse comprises a nucleotide sequence thatencodes a FR1, FR2, and FR3 sequence of an IgG heavy chain that lacks afunctional CH1 region, that is at least 80% identical to an FR1, FR2,and FR3 from a human germline nucleotide sequence of a 1-8, 1-18, or1-69 human immunoglobulin heavy chain variable region gene segment;wherein the FR1+FR2+FR3 sequence of the modified mouse is optimallyaligned with the recited human germline sequence without regard to thesequence of the CDRs of the mouse and human sequences (i.e., optimallyaligning the FRs while not considering the identities of amino acids ofany CDRs in the comparison). In specific embodiments, the FR1, FR2, andFR3 are about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a humangermline nucleotide sequence of a FR1+FR2+FR3 of a heavy chain variableregion gene segment that is a 1-8, 1-18, or 1-69 gene segment.

In one embodiment, the mouse further comprises a FR4 that is at least80% identical to a FR4 formed by a human D6-19/J6 rearrangement, aD6-7/J4 rearrangement, a D4-4/J4 rearrangement, a D6-6/J2 rearrangement,a D3-16/J6 rearrangement, a D6-6/J4 rearrangement, and a D1-7/J4rearrangement. In specific embodiments, the FR4 is about 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to an FR4 formed by the aforementionedD/J rearrangement.

In one embodiment, the mouse comprises a nucleotide sequence encoding aFR1 whose amino acid sequence differs by no more than 1, no more than 2,no more than 3, no more than 4, or no more than 5 amino acids from a FR1encoded by a germline sequence of human heavy chain variable region genesegment selected from V1-8, V1-18, and V1-69. In a specific embodiment,the nucleotide sequence encoding the FR1 is a rearranged sequenceoperably linked to a sequence encoding an IgG constant region that lacksa functional CH1 sequence.

In one embodiment, the mouse comprises a nucleotide sequence encoding aFR2 whose amino acid sequence differs by no more than 1, no more than 2,no more than 3, no more than 4, or no more than 5 amino acids from a FR2encoded by a germline sequence of human heavy chain variable region genesegment selected from V1-8, V1-18, and V1-69. In a specific embodiment,the nucleotide sequence encoding the FR2 is a rearranged sequenceoperably linked to a sequence encoding an IgG constant region that lacksa functional CH1 sequence.

In one embodiment, the mouse comprises a nucleotide sequence encoding aFR3 whose amino acid sequence differs by no more than 1, no more than 2,no more than 3, no more than 4, no more than 5, no more than 6, no morethan 7, no more than 8, no more than 9, no more than 10, or no more than11 amino acids from a FR3 encoded by a germline sequence of human heavychain variable region gene segment selected from V1-8, V1-18, and V1-69.In a specific embodiment, the nucleotide sequence encoding the FR3 is arearranged sequence operably linked to a sequence encoding an IgGconstant region that lacks a functional CH1 sequence.

In one embodiment, the mouse comprises a nucleotide sequence encoding aFR4 whose amino acid sequence differs by no more than 1, no more than 2,or no more than 3 amino acids from a FR4 amino acid sequence encoded bya rearrangement of a human D6-19/J6, a D6-7/J4, a D4-4/J4, a D6-6/J2, aD3-16/J6, a D6-6/J4, and a D1-7/J4. In a specific embodiment, thenucleotide sequence encoding the FR4 is a rearranged sequence operablylinked to a sequence encoding an IgG constant region that lacks afunctional CH1 sequence.

In one embodiment, the mouse comprises a nucleotide sequence encoding aheavy chain CDR3 derived from a human heavy chain D region gene segment(hDH). In one embodiment, the hDH is selected from D1-7, D3-16, D4-4,D6-6, D6-7, and D6-19.

In one embodiment, the mouse comprises a nucleotide sequence encoding aheavy chain CDR3 derived from a human heavy chain joining gene segment(JH). In a specific embodiment, the JH is selected from J2, J4, and J6.

In one embodiment, the mouse comprises a heavy chain CDR3 encoded by anucleotide sequence derived from a human DH and a human JHrearrangement. In a specific embodiment, the CDR3 is derived from aD1-7/J4, D3-16/J6, D4-4/J4, D6-6/J2, D6-6/J4, D6-7/J4, or a D6-19/J6rearrangement.

In one embodiment, the mouse comprises a replacement of an endogenousmVR gene segment with an hVR gene segment. In a specific embodiment, thereplacement of the mVR gene segment with the hVR gene segment is on thesame allele as the modified heavy chain constant region. In anotherspecific embodiment, the replacement of the mVR gene segment with thehVR gene segment is on a different allele than the modified heavy chainconstant region.

In one embodiment, 90-100% of mVR gene segments are replaced with atleast one hVR gene segment. In a specific embodiment, all orsubstantially all of the endogenous mVR gene segments are replaced withat least one hVR gene segment. In one embodiment, the replacement iswith at least 18, at least 39, or at least 80 or 81 hVR gene segments.In one embodiment, the replacement is with at least 12 functional hVRgene segments, at least 25 functional hVR gene segments, or at least 43functional hVR gene segments.

In one embodiment, the genetically modified mouse comprises a transgenethat comprises at least one unrearranged hVR gene segment, at least oneunrearranged human D segment, at least one unrearranged human J segment,and at least one human heavy chain constant sequence. In one embodiment,the endogenous mouse heavy chain variable region and kappa light chainvariable region loci are functionally silenced. In a specificembodiment, the mouse is capable of trans-switching to produce achimeric human/mouse antibody comprising a human heavy chain variabledomain contiguous with a mouse IgG sequence that lacks a functional CH1domain and, optionally, lacks a hinge region of the IgG that lacks thefunctional CH1 domain. In a specific embodiment, the transgene furthercomprises an IgG sequence that lacks a CH1 domain, and optionallycomprises an IgM having a functional CH1 domain. In a further specificembodiment, the IgG sequence lacks a hinge region.

In one embodiment, the mouse comprises a first heavy chain variableregion allele and a second heavy chain variable region allele, whereinthe first allele and the second allele are both from the same mousestrain. In one embodiment, the first allele is from a first mouse strainand the second allele is from a second mouse strain. In one embodiment,one allele of the first and the second alleles comprises a replacementof an mVR with at least one hVR. In another embodiment, both allelescomprise a replacement of an mVR with at least on hVR.

In one aspect, a genetically modified mouse is provided, wherein themouse expresses an IgM that comprises a CH1 domain, and the mouseexpresses an IgG that lacks a functional CH1 domain or that expresses anIgG that lacks both a functional CH1 domain and that lacks a functionalhinge region.

In one embodiment, the IgG is an IgG1.

In one embodiment, the mouse expresses four IgGs that are: a modifiedIgG1 and a wild-type IgG3, IgG2a, and IgG2b. In another embodiment, themouse expresses no more than two IgGs that are: a modified IgG1 and awild-type IgG3. In a specific embodiment the mouse expresses heavy chainisotypes that are: a wild-type IgM, a wild-type IgD, a wild-type IgG3, amodified IgG1, a wild-type IgG2a, a wild-type IgG2b, a wild-type IgA,and a wild-type IgE. In another specific embodiment, the mouse expressesheavy chain isotypes that are: a wild-type IgM, a wild-type IgD, awild-type IgG3, a modified IgG1, a wild-type IgA, and a wild-type IgE.In various embodiments, the modification of the IgG1 comprises adeletion of a CH1 domain and, optionally, a deletion of a hinge region.

In one embodiment, the mouse is from a strain selected from 129,C56BL/6, a mixed 129×C57BL/6.

In one aspect, a mouse that expresses a heavy chain antibody isprovided, wherein the heavy chain antibody consists essentially of adimeric heavy chain, wherein the heavy chain lacks a functional CH1domain or lacks both a functional CH1 domain and a functional hingeregion, the heavy chain comprises a mammalian heavy chain variabledomain that comprises a sequence that is not identical to a mammalianheavy chain variable domain encoded by a germline variable region gene,and the heavy chain comprises a human or mouse CH2 domain and a human ormouse CH3 domain, wherein the mouse expresses a wild-type human or mouseIgM.

In one embodiment, the mouse comprises a functional immunoglobulin lightchain gene locus.

In one embodiment, wherein the mammalian heavy chain variable domain isa human or mouse heavy chain variable domain.

In one embodiment, the heavy chain antibody consists essentially of adimeric heavy chain lacking a functional CH1 domain and lacking afunctional hinge region, wherein the heavy chain comprises a humanvariable domain that comprises at least one somatic mutation andcomprises a CH2 domain and a CH3 domain. In a specific embodiment, theCH2 domain and the CH3 domain are independently selected from mouse andhuman domains. In a specific embodiment, both the CH2 and the CH3 domainare human; in another embodiment, both the CH2 and the CH3 domain aremouse.

In one aspect, a heavy chain antibody is provided, wherein the heavychain antibody comprises a heavy chain comprising a non-camelid variabledomain and a heavy chain constant region lacking a CH1 domain.

In one embodiment, the heavy chain antibody further lacks a hingeregion.

In one embodiment, the heavy chain antibody comprises a constant regionthat consists essentially of a hinge region, a CH2 domain, and a CH3domain. In another embodiment, the heavy chain antibody comprises aconstant region that consists essentially of a CH2 domain and a CH3domain.

In one embodiment, the non-camelid variable domain is a somaticallymutated human or mouse heavy chain variable domain obtained from an IgM-or an IgG-encoding nucleotide sequence of a B cell from a mouse or agenetically modified mouse comprising a human heavy chain variableregion gene segment. In a specific embodiment, the mouse comprises ahumanized heavy chain variable region gene segment. In anotherembodiment, the mouse comprises a replacement of the endogenous mouseheavy chain variable region gene segment locus with at least one humanvariable region gene segment. In another embodiment, the mouse comprisesa replacement of the endogenous mouse heavy chain locus with at leastone human variable gene segment, at least one human D gene segment, andat least one human J gene segment. In a specific embodiment, theendogenous mouse immunoglobulin variable region locus is all orsubstantially all replaced with a human immunoglobulin variable regionlocus comprising a plurality of human V, D, and J gene segments.

In one embodiment, the non-camelid variable domain is a human or a mousevariable domain. In another embodiment, the non-camelid variable domainis a human or a mouse variable domain comprising one or more camelizingmodifications. In a specific embodiment, the camelizing modification isselected from L11S, V37F, G44E, L45C, L45R, and W47G (Kabat numbering).In a specific embodiment, the camelizing modification is selected fromV37F, G44E, and L45C. In a specific embodiment, the heavy chain variabledomain comprises a complementarity determining region 3 (CDR3) thatcomprises two cysteines.

In one embodiment, the heavy chain antibody comprises a dimer of a firstheavy chain comprising a first heavy chain variable domain and a secondheavy chain comprising a second heavy chain variable domain, whereineach of the first and the second heavy chains lacks a CH1 domain (orlacks a CH1 domain and a hinge region). In one embodiment, the humanvariable domain of the first heavy chain of the dimer binds a firstepitope, and the human variable domain of the second heavy chain of thedimer binds a second epitope, wherein the first and the second epitopeare not identical. In a specific embodiment, the heavy chain variabledomains of the first and the second heavy chains comprise human heavychain variable domains and/or human heavy chain FR regions as describedherein.

In one aspect, a genetically modified non-human cell is provided,wherein the genetic modification comprises a deletion of an IgG CH1domain and the cell expresses a functional IgM. In a specificembodiment, the cell comprises an IgM gene comprising a sequenceencoding a CH1 domain.

In one embodiment, the cell is selected from a non-human ES cell, apluripotent cell, and a totipotent cell. In a specific embodiment, thenon-human ES cell is selected from a mouse ES cell and a rat ES cell.

In one aspect, a genetically modified non-human embryo is provided,wherein the genetic modification comprises a modification as describedherein. In one embodiment, the genetic modification comprises a deletionof an IgG CH1 domain and the non-human embryo expresses a functionalIgM. In a specific embodiment, the non-human embryo comprises an IgMgene comprising a CH1 domain.

In one embodiment, the non-human embryo is a mouse embryo or a ratembryo.

In one aspect, a non-human embryo comprising a donor cell is provided,wherein the donor cell is genetically modified, and wherein the geneticmodification is a modification as described herein. In one embodiment,the genetic modification comprises a deletion of an IgG CH1 domain andthe cell comprises an IgM gene comprising a CH1 domain.

In one embodiment, the non-human embryo is a mouse embryo or a ratembryo, and the donor cell is a mouse ES cell or a rat ES cell,respectively.

In one aspect, a DNA construct is provided, wherein the DNA constructcomprises (a) a mouse homology arm homologous to a first sequence 5′ andimmediately adjacent to the start of an IgG CH1 region; (b) a marker ordrug selection cassette; and, (c) a homology arm homologous to a secondsequence 3′ and immediately adjacent to the end of an IgG CH1 region or,alternatively, a homology arm homologous to a second sequence 3′ andimmediately adjacent to the end of an IgG hinge region.

In one aspect, a method for making an antibody that lacks a CH1 domainis provided, comprising: (a) immunizing a non-human animal as describedherein that lacks a functional CH1 domain in an IgG or lacks afunctional CH1 domain and lacks a functional hinge region in the IgG,wherein the mouse expresses an IgM that comprises a functional CH1domain; (b) maintaining the non-human animal under conditions sufficientfor the non-human animal to make an antibody; (c) identifying anantibody made by the mouse that lacks a functional CH1 domain or thatlacks a functional hinge region; and, (d) isolating from the mouse theantibody, a cell that makes the antibody, or a nucleotide sequence thatencodes a sequence of the antibody.

In one embodiment, the non-human animal comprises a functionalimmunoglobulin light chain gene locus.

In one aspect, a method for humanizing a mouse heavy chain antibody isprovided, comprising immunizing a genetically modified mouse that makesheavy chain antibodies with an antigen of interest, allowing the mouseto mount an immune response, identifying a mouse VH region of the mousethat is encoded in a B cell of the mouse, wherein the B cellspecifically binds the antigen of interest, and humanizing the VHregion.

In one embodiment, the genetically modified mouse that makes heavy chainantibodies is a mouse as described herein. In one embodiment, the mousecomprises at least one mVR gene segment operably linked to a heavy chainconstant locus that comprises an intact IgM gene and that comprises anIgG gene that lacks a CH1 domain or that lacks a CH1 domain and lacks ahinge domain. In one embodiment, the IgG gene is an IgG1 gene. In oneembodiment, the IgG gene is selected from IgG1, IgG2A, IgG2B, IgG3, anda combination thereof.

In one embodiment, the method further comprises cloning a nucleotidesequence encoding the humanized VH region onto a nucleotide sequence ofa human immunoglobulin constant region.

In one embodiment, the mouse mVR gene segment is from a mouse VH genefamily selected from VH1 and VH14, and the humanization comprisesreplacing a mouse framework of VH1 or VH14 with a framework from a humanVH1 gene. In one embodiment, the human VH1 gene is selected from 1-2,1-3, 1-8, 1-17, 1-18, 1-24, 1-45, 1-46, 1-58, and 1-69. In specificembodiments, the mVR gene is a 1-58 gene and the human gene is a 1-18gene; the mVR gene is a 1-26 gene and the human gene is a 1-2 gene; themVR gene is a 1-50 gene and the human gene is a 1-46 gene; the mVR geneis a 1-17 gene and the human gene is a 1-2 gene; the mVR gene is a 1-42gene and the human gene is a 1-2 gene; the mVR is a 14-1 gene and thehuman gene is a 1-2 gene; or the mVR is a 14-2 gene and the human geneis a 1-2 gene.

In one embodiment, the mVR gene segment is from a mouse VH gene selectedfrom a VH4, VH5, VH6, VH7, VH10, VH11, and VH13 gene, and thehumanization comprises replacing a mouse framework with a framework froma human VH3 gene. In one embodiment, the human VH3 gene is selected from3-7, 3-9, 3-11, 3-13, 3-15, 3-16, 3-20, 3-21, 3-23, 3-30, 3-33, 3-35,3-38, 3-43, 3-48, 3-49, 3-53, 3-64, 3-66, 3-72, 3-73, and 3-74. In aspecific embodiment, the mVR gene is a 7-1 gene and the human gene is a3-72 gene; the mVR gene is a 3-6 gene and the human gene is a 4-59 gene;the mVR gene is a 5-6 gene and the human gene is a 3-21 gene.

In one embodiment, the mVR gene segment is from a mouse VH gene familyselected from VH3 and VH12, and the humanization comprises replacing amouse framework with a framework from a human VH4 gene. In oneembodiment, the human VH4 gene is selected from 4-4, 4-28, 4-31, 4-34,4-39, 4-59, and 4-61.

In one embodiment, the mVR gene segment is from a mouse VH4 gene family,and the humanization comprises replacing a mouse VH4 framework with aframework from a human VH6 gene. In one embodiment, the human VH6 geneis 6-1.

In one embodiment, the mVR gene segment is from a mouse VH9 gene family,and the humanization comprises replacing a mouse VH9 framework with aframework from a human VH gene of the human VH7 family. In oneembodiment, the human VH gene is selected from 7-4-1 and 7-81.

In one embodiment, the humanization further comprises making one or moreconservative or non-conservative substitutions, one or more deletions,and/or one or more insertions in a mouse CDR such that the mouse CDRcorresponds more closely to a human CDR.

In one embodiment, the humanization further comprises making one or moreconservative or nonconservative substitutions, one or more deletions,and/or one or more insertions in a human framework such that the humanframework corresponds more closely to the mouse framework.

In one aspect, a genetically modified mouse is provided that comprises afunctional immunoglobulin light chain gene, wherein the mouse expressesa heavy chain antibody that lacks a light chain and that lacks a CH1region or that lacks a CH1 region and a hinge region.

In one embodiment, the mouse comprises an immunoglobulin gene that lacksa sequence encoding a CH1 region, or lacks a sequence encoding a hingeand a CH1 region. In one embodiment, the immunoglobulin gene that lacksthe sequence is one or more heavy chain constant genes. In a specificembodiment, the immunoglobulin gene that lacks the sequence is selectedfrom an IgG1, IgG2a, IgG2b, and IgG3 gene. In a specific embodiment, themouse comprises an IgM gene with a CH1 region, and/or a hinge region,and/or a CH1 region and hinge region.

In one embodiment, the antibody is expressed in response to an antigen,and the antibody specifically binds the antigen.

In one embodiment, the antibody comprises a mouse VH domain. In aspecific embodiment, the mouse VH domain comprises a mouse VH genesegment selected from 1-26, 1-42, 1-50, 1-58, 1-72, 3-6, 5-6, 7-1, 14-1,and 14-2.

In one embodiment, the antibody comprises a human VH domain. In aspecific embodiment, the human VH domain comprises a sequence derivedfrom a human VH gene segment selected from 1-2, 1-18, 1-46, 3-21, 3-72,and 4-59.

In one aspect, a genetically modified mouse is provided that expresses abinding protein that consists essentially of two IgG1 heavy chains thateach lack a CH1 domain, wherein the mouse expresses an IgM thatcomprises a CH1 region, and wherein the mouse is incapable of expressingfrom its genome an mRNA that comprises a nucleotide sequence encoding aCH1 domain of an IgG1.

In one embodiment, the immunoglobulin heavy chains that each lack a CH1domain consist essentially of, from N-terminal to C-terminal, a human ormouse heavy chain immunoglobulin variable region, optionally a hingeregion, a mouse CH2 region, and a mouse CH3 region. In a specificembodiment, the heavy chain immunoglobulin variable region is a humanvariable region, a hinge region is present, and the mouse comprises afunctional immunoglobulin light chain gene locus.

In one aspect, a mouse is provided that expresses a heavy chain antibodythat lacks a light chain and that lacks a CH1 region in whole or inpart, wherein the mouse expresses a B cell receptor on a B cell, whereinthe B cell receptor on its surface displays a binding molecule thatcomprises an immunoglobulin heavy chain variable domain fused directlyto an immunoglobulin hinge region or fused directly to a CH2 region,wherein the binding molecule lacks a CH1 region. In one embodiment, thebinding molecule comprises an IgG1 CH2 and CH3 region.

In one aspect, a method for making a heavy chain antibody is provided,comprising immunizing a mouse with an antigen of interest, wherein themouse comprises an IgG gene that lacks a sequence encoding a CH1 region,wherein the mouse comprises an intact IgM constant region gene, allowingthe mouse to mount an immune response against the antigen of interest,and isolating from the mouse a cell or protein that specificallyrecognizes the antigen of interest, wherein the cell or proteincomprises a heavy chain antibody that lacks a CH1 domain and that lacksa cognate light chain and that specifically binds the antigen ofinterest.

In one embodiment, the mouse comprises a functional light chain gene. Inone embodiment, the mouse comprises a functional light chain geneselected from lambda, kappa, and a combination thereof.

In one embodiment, the mouse comprises a replacement of all orsubstantially all mouse heavy chain V, D, J gene segments with one ormore human V, D, J gene segments.

In one embodiment, the IgG gene that lacks the sequence encoding a CH1is selected from an IgG1, IgG2a, IgG2b, IgG3, and a combination thereof.

In one embodiment, the IgG gene that lacks the CH1 sequence is IgG1, andthe mouse lacks a gene encoding IgG2a, IgG2b, IgG3, or a combinationthereof. In one embodiment, the IgG gene that lacks the CH1 sequence isIgG2a, and the mouse lacks a gene encoding IgG1, IgG2b, IgG3, or acombination thereof. In one embodiment, the IgG gene that lacks the CH1sequence is IgG2b, and the mouse lacks a gene encoding IgG1, IgG2a,IgG3, or a combination thereof. In one embodiment, the IgG gene thatlacks the CH1 sequence is IgG3, and the mouse lacks a gene encodingIgG1, IgG2a, IgG2b, or a combination thereof.

In one embodiment, the mouse comprises a B cell that bears on itssurface a B cell receptor, wherein the B cell receptor comprises arearranged heavy chain VDJ that binds the antigen of interest, andwherein the B cell receptor comprises an IgM that comprises a CH1region, and wherein the IgM comprises a light chain. In one embodiment,the light chain is VJ rearranged. In a specific embodiment, the lightchain is a kappa or a lambda light chain that is cognate with therearranged heavy chain VDJ that binds the antigen of interest.

In one aspect, a mouse heavy chain antibody, human heavy chain antibody,or chimeric human/mouse heavy chain antibody made in a mouse accordingto the invention is provided.

In one aspect, a mouse heavy chain antibody, human heavy chain antibody,chimeric human/mouse heavy chain antibody, or humanized heavy chainantibody made using a heavy chain variable region nucleotide sequence orfragment thereof made in a mouse according to the invention is provided.

Other embodiments are described and will become apparent to thoseskilled in the art from a review of the ensuing detailed description.

DETAILED DESCRIPTION

The invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. Theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, particular methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference in their entirety.

CH1 Domains and Antibody Production

Genetically modified non-human animals are provided that make antibodiesthat lack a CH1 domain, including heavy chain antibodies, i.e.,antibodies that lack light chains. The genetically modified non-humananimals comprise a genetic modification that includes a lack of afunctional immunoglobulin heavy chain domain (a CH1 domain), e.g., anIgG1 CH1 domain, and in some embodiments a further modificationcomprising a deletion of a hinge region in the immunoglobulin heavychain that lacks the functional CH1 domain, wherein the non-human animalexpresses a functional IgM. Other modifications include renderingisotypes other than IgG1 and IgM to be nonfunctional, e.g., makingdeletions in genes, or deletions of genes, for IgD, IgG3, IgG2a, IgG2b,IgA, and IgE. Genetically modified non-human embryos, cells, andtargeting constructs for making the non-human animals, non-humanembryos, and cells are also provided.

Efforts at making genetically modified cells that can make heavy chainantibodies (i.e., antibodies that lack a light chain) have focused onmimicking heavy chain antibodies in other species, e.g., in camelids andcertain fish. This approach has been used to genetically modify a mouseES cell to delete CH1 domains in immunoglobulin constant region genes ofIgMs and IgGs, and also to introduce heavy chain variable regions intothe ES cell that are camelid, or camelized VHH or VHH-like). Thedeletion of IgM and IgG CH1 domains is undertaken presumably to preventformation of endogenous, natural antibodies to compete with camelizedantibody formation from a genetically modified locus. The addition ofVHH gene segments is undertaken presumably to mimic heavy chain antibodyformation in combination with the CH1 deletion. Heavy chain antibodiesfrom such animals will contain the VHH gene segment. VHH gene segmentsare presumably thought to be necessary for the proper expression of aheavy chain antibody, since in vitro studies indicate that non-camelidVH domains do not satisfactorily form expressible heavy chain antibodieswhen present in heavy chains lacking a CH1 domain.

In camelids, however (and in some cartilaginous fish), genes are presentthat include CH1 domains, or CH1-like domains. VHH-containing antibodiesthat lack CH1 domains are believed to result from RNA splicing or fromrearrangement of DNA sequences that can encode a CH1 region. Thus, evencamelids have retained DNA sequences encoding CH1 regions. Becausehumans (under some circumstances) can make heavy chain antibodieslacking a CH1 region in whole or in part (e.g., in human heavy chaindisease), it might be possible to compel non-camelids, such as mice, toform heavy chains lacking a CH1 region under a given set ofcircumstances. This approach relies upon not disturbing the germlinestructure of a CH, but instead rendering the animal's light chain locusnonfunctional. This approach assumes that with a nonfunctional lightchain locus those heavy chains that require a cognate light chain forexpression (e.g., full-length heavy chains having CH1 regions) are notmade due to the lack of any kappa or lambda light chain, such that onlythose heavy chains that can express and secrete without a light chain(i.e., heavy chains lacking a CH1 region) will be expressed andsecreted. The approach relies upon the absence of functional kappa orlambda gene segments that can rearrange to form a functional light chaingene, and on the absence of any functional rearranged light chain gene,and thus requires a genetic manipulation (e.g., a knockout) to destroyfunctionality of both germline light chain loci. The approach reliesupon “natural” processes leading to non-use of the endogenous CH1nucleotide sequence, and that the “natural” process of CH1 silencingoccurs in class switching. There does not appear to be any possibilityof using such a process in any animal that contains a functional lightchain gene. Furthermore, it appears that the “natural” process includesthe synthesis of large amounts of normal RNA, i.e., RNA that includes aregion encoding a CH1 region.

Compositions and methods are provided for making a mouse that makes anantibody that lacks an immunoglobulin CH1 domain (and optionally a hingeregion), including heavy chain antibodies, and including antibodies thatcomprise VH domains (e.g., mouse or human VH domains). The methodsinclude selectively rendering an endogenous non-IgM CH1 region to benonfunctional (e.g., by a deletion of a sequence of a CH1 domain), andemploying either unrearranged endogenous mouse variable region (mVR)gene segments or unrearranged human variable region (hVR) gene segmentsat the endogenous mouse variable region locus to make a chimerichuman/mouse antibody in a mouse. The deletion of the CH1 domain is madein one or more IgG genes, but not in an IgM gene. The approachselectively renders one or more IgG CH1 domains nonfunctional whileretaining a functional IgM. In addition to a deletion of the one or moreIgG CH1 domains, a further embodiment provides for deleting or renderingnonfunctional the hinge region of the IgG(s) in which the CH1 domain isdeleted or rendered nonfunctional.

The IgG CH1 deletion approach employs a relatively conservativedisruption in natural B cell development in the animal, because not allIg isotypes of the genetically modified non-human animal will exhibit anonfunctional CH1 or a deletion of the CH1 domain (and, optionally,hinge). Thus, the CH1 modification does not occur in IgM molecules andthus does not affect those steps in early B cell development that dependon an IgM having a functional CH1. Because the IgM is not modified,animals bearing one or more deletions of the CH1 domain of an IgG (andoptionally a hinge region of the IgG), but not an the CH1 domain of anIgM, should be able to process a satisfactorily large repertoire ofvariable regions in clonal selection steps prior to presentation of thevariable domain in the context of an IgG. Thus in various embodiments,any deleterious affect of the genetic modification(s) on the diversityof variable regions available for use in a heavy chain antibody shouldnot negatively impact the pool of variable regions available forselection in an IgG context. Further, where the CH1 sequence that isrendered nonfunctional (e.g., deleted) in the germline is an IgG1, themouse will lack the ability to make any RNA that encodes a CH1 domain.

Genetically modifying a non-human animal to render a CH1 domain or a CH1domain and a hinge region of one or more IgG isotypes nonfunctional mayresult in a mouse that is able to select, from a full or substantiallyfull repertoire of VH regions, a suitable VH region to express in aheavy chain antibody. Selectively modifying IgG isotypes (but not IgM)avoids a potential reduction in the number of VH regions that surviveselection due to a lack of a CH1 domain or a lack of a CH1 domain inIgM. Thus, a fuller repertoire of VH regions is available for selectionin the context of an IgG (that lacks a CH1 domain or that lacks a CH1domain and that lacks a hinge region). Thus, selection of a VH domain ina genetically modified mouse in accordance with the invention does notdepend, e.g., on which VH domain might help overcome early IgM-dependentB cell developmental hurdles that are due to modified IgM structures.Instead, early IgM-dependent steps should occur as normal, resulting ina large repertoire of heavy chains available for selection as to theirsuitability to express in the context of an IgG that lacks a CH1 domainor that lacks a CH1 domain and lacks a hinge region.

Thus, in various embodiments, a genetically modified mouse in accordancewith the invention should maintain functional IgM expression, whichshould provide an opportunity for a more natural clonal selectionprocess. For example, with a functional IgM (e.g., an IgM that does notlack a CH1 domain), both surrogate light chain and the cognate lightchain will be able to associate through the IgM's CH1 domain andparticipate in selection processes in early B cell development. In agenetically modified mouse in accordance with the invention, it isbelieved that class switching to an IgG isotype is the first selectionstep where any selection of heavy chain variable domains that can beexpressed in the context of a constant domain lacking a functional CH1domain or lacking a functional CH1 domain and a functional hinge isencountered.

IgM in B Cell Development

Although observations in camelids, certain fish, and in pathologicalconditions reveal that under some circumstances an antibody that lacks aCH1 domain of its heavy chain constant region can be expressed in theabsence of a cognate light chain, normal development ofantibody-producing B cells generally requires the presence of a CH1domain. All heavy chain isotypes, including IgM, comprise a CH1 domain.Both the surrogate light chain and a cognate light chain are believed tointeract with a given heavy chain through the heavy chain's CH1 domainin the context of an IgM. To the extent that development of heavy chainantibodies depends upon structural integrity or functionality of an IgMisotype heavy chain, disruption of the IgM's structural integrity orfunction would be undesirable.

Normal development of antibodies requires that antibodies survivethroughout a multiplicity of complex selection schemes that result inthe survival and ultimate expression of functional and usefulantibodies. Disruptions in antibody structure can prove deleterious tothe survival and ultimate expression of an antibody to the extent thatthe structural disruption results in the inability of the antibody toeffectively compete and evolve to the satisfaction of one or more ofnature's antibody selection schemes.

Early in antibody development, antibody heavy chains undergo a selectionprocess wherein nature chooses, through a variety of selection schemes,suitable heavy chains to undergo further selection to eventually formfunctional and affinity-matured antibodies. Antibody heavy chainsexpressed from recombined heavy chain gene segments in progenitor Bcells (or, pro-B cells) are normally paired with a surrogate light chainfor presentation on the surface of the pro-B cell in an IgM isotype toform a structure (which includes other co-receptors) referred to as apre-B cell receptor, or pre-BCR. Once the pre-BCR is presented on thecell surface, the pre-BCR is believed to signal its appropriateformation of the complex to the cell, effectively instructing the cellthat the heavy chain has passed this early selection step. Thus the cellis informed that the heavy chain may undergo further selection. If theheavy chain contains a defect that is deleterious to the formation of apre-BCR when presented in the context of an IgM and a surrogate lightchain, the cell will undergo apoptosis. If the cell undergoes apoptosis,the usefulness, or contribution to diversity, of the heavy chainvariable region of the heavy chain will be lost. Thus, a very early stepin antibody selection requires presentation of the heavy chain togetherwith a surrogate light chain in the context of an IgM isotype. Thesurrogate light chain is believed to interact with IgM at least in partthrough IgM's CH1 domain. A failure or disruption in antibody structureat this early juncture (e.g., a nonfunctional CH1 domain) can result inclonal selection failure, loss of the pro-B cell that expresses theheavy chain, and loss of the possibility of employing the particularheavy chain variable domain in a useful antibody.

Once the cell bearing the pre-BCR passes this selection step, the nextselection step requires that the heavy chain be paired with a cognatelight chain (e.g., either kappa or lambda in mice and humans). Thepaired heavy chain/cognate light chain structure is again presented onthe surface of the cell, now a naive pre-B cell, in the context of anIgM isotype through the IgM's CH1 domain. This complex on the surfaceresults in a functional, membrane-bound, B cell receptor (BCR). This BCRis believed to signal to the cell that the heavy chain is suitable forfurther selection, and that the cell may now commit to expressing thisparticular light chain and proceed to further B cell maturation steps,including affinity maturation and class switching. If the heavy chaincontains a defect that is deleterious to the formation of a BCR whenpresented in the context of an IgM and its cognate light chain, the cellwill undergo apoptosis. If the cell undergoes apoptosis, the usefulness,or contribution to diversity, of the heavy chain variable region of theheavy chain will be lost. Thus, a very early step in antibody selectionrequires presentation of the heavy chain together with a surrogate lightchain in the context of an IgM isotype. Again, a failure or disruptionin antibody structure (e.g., a non-functional CH1 domain) at this earlyjuncture can result in clonal selection failure and concomitant loss ofthe pre-B cell that expresses the heavy chain.

Having survived selection thus far, the pre-B cell that presents theheavy chain paired with its cognate light chain in the IgM context thenundergoes a maturation process that ultimately results in classswitching and further selection mechanisms in which the heavy chain andcognate light chain are presented on the B cell surface in the contextof an IgG isotype. It would be at this step that any selection of IgGheavy chains that lack a CH1 domain or that lack a CH1 domain and ahinge region would occur. In animals according to the invention, it isbelieved that a normal repertoire of heavy chain variable regions wouldbe available for selection based upon whether the variable domain wouldsurvive to be expressed in an IgG heavy chain that lacks a CH1 domain orthat lacks a CH1 domain and a hinge region. In contrast, mice that haveimpaired IgMs would likely not present a full repertoire of heavy chainvariable regions, since only those variable regions capable of survivingselection in the context of an impaired IgM would be available for classswitching.

Thus, an animal lacking a functional IgM may experience a markedreduction in the ability to make a B cell population followingrearrangement of otherwise suitable heavy chain variable gene segments.In such a case, even where an ample supply of heavy chain variableregions is available (i.e., the animal has a suitable number of heavychain variable region gene segments capable of rearranging), asatisfactory population of B cells that display a desirable degree ofdiversity may not form because of an IgM impairment that mitigatesagainst survival of a heavy chain during the selection process.

Heavy Chain Antibody Production with a Functional IgM Gene

A suitable number of rearranged heavy chain variable regions that caneffectively survive selection when presented during B cell developmentin the context of an IgM is desirable to be maintained in order togenerate sufficient diversity to make antibodies by immunizing anon-human animal with an immunogen of interest. Thus, a geneticallymodified non-human animal that comprises a nonfunctional CH1 domain or anonfunctional CH1 domain and a nonfunctional hinge region in animmunoglobulin heavy chain should not comprise a CH1 deletion in bothIgM alleles.

In some embodiments, it is not desirable to delete CH1 domains of all Igisotypes in order to make a heavy chain antibody in a geneticallymodified animal. Thus, methods and compositions are provided for makinga heavy chain antibody in a genetically modified non-human animal bydisabling, deleting, or otherwise rendering non-functional a nucleotidesequence encoding a CH1 domain or fragment thereof of an IgG (and insome embodiments also disabling, deleting, or otherwise renderingnonfunctional a hinge region of the IgG) while allowing other isotypes(e.g., IgM) to retain functional CH1 domains. It is believed thatfunctionality of other isotype CH1 domains (other than one or moreselected IgG CH1 domains) results in a B cell development process thatdoes not disrupt or substantially disrupt developmental steps in whichthe heavy chain variable domain is presented in the context of a non-IgGisotype, e.g., in an IgM isotype. Thus disruption of, e.g.,IgM-dependent steps during B cell development is relatively minimized.Without limitation as to the invention (which is described by theclaims) the inventors propose that minimalizing disruption of earlyselection steps associated with presentation of the heavy chain variabledomain in an IgM context will result in more cells that bear the heavychain variable regions surviving to undergo class-switching to an IgGisotype and selection in the context of an IgG that lacks a functionalCH1 domain or that lacks a functional CH1 domain and lacks a functionalhinge region.

Accordingly, a genetically modified non-human animal is provided, alongwith methods and compositions for making the animal, wherein the geneticmodification results in lack of a functional CH1 domain (in a furtherembodiment lack of a functional hinge region) in an Ig domain that isnot an IgM domain. In various embodiments, a sequence encoding CH1 orthe CH1 and the hinge region (or a substantially functional portionthereof) are deleted in the genome of the genetically modified animal.The genetically modified non-human animal is useful in making heavychain antibodies (i.e., antibodies that lack a light chain), includingfully human antibodies (in a mouse genetically modified to include humanimmunoglobulin genes) and chimeric human/mouse antibodies (e.g., in amouse genetically modified to include human variable region genesegments, D regions, and J regions, or in a mouse having a humantransgene capable of trans-switching to a genetically modified IgGisotype that lacks a functional CH1 domain or that lacks a functionalCH1 domain and lacks a functional hinge region).

Heavy Chain Antibodies

Antibodies are useful as human therapeutics. Heavy chain antibodies,i.e., antibodies that lack a light chain, are also useful as humantherapeutics. Because heavy chain antibodies lack a light chain, theyare smaller and thus expected to exhibit better tissue penetration thanantibodies that contain light chains, yet have a similar or morefavorable pharmacokinetic profile and yet retain similar effectorfunction as compared to a conventional antibody. Because they aresmaller, heavy chain antibodies are also capable of administration at ahigher dose in a given volume. A frequent method of administeringantibodies is by subcutaneous injection, and a reduction inadministration volume for a given dosage of antibody can providebenefits to patients and avoid complications and pain due tosubcutaneous injections of large volumes.

Another advantage of heavy chain antibodies is the ability to makebispecific antibodies by heterodimerizing heavy chains with specificityfor two different epitopes in a single therapeutics. Because heavy chainantibodies lack a light chain, they are particularly suited for makingbispecific antibodies since there is no requirement to engineer a commonlight chain that would not interfere with binding affinity orspecificity of either heavy chain but also enable suitable expression ofthe bispecific antibody.

The genetically modified animals of the invention can be used to make awide variety of heavy chain antibodies. The genetic modificationsdescribed herein can be made, e.g., in any suitable mouse strain. Themouse strain can have any genetic background suitable for making a heavychain antibody of choice. Some genetic backgrounds that encompassparticular embodiments are provided below.

The genetically modified animal can be a mouse comprising a geneticmodification in accordance with the invention and one or moreunrearranged human variable region gene segments, one or moreunrearranged D region gene segments, and one or more unrearranged Jregion gene segments replacing an endogenous mouse heavy chain variableregion locus. In such a mouse, the humanized variable region locus iscapable of recombining to form a rearranged variable region geneupstream of endogenous mouse constant domain sequences (wherein one ormore of the immunoglobulin constant region genes is modified asdescribed herein). The mouse would thus be capable of making a chimerichuman variable/mouse constant heavy chain antibody. Upon exposure to animmunogen of interest, the mouse would be capable of generating a heavychain antibody in accordance with the invention that is affinity maturedand capable of specifically binding an epitope of the immunogen ofinterest.

The genetically modified animal can be a mouse comprising an endogenousmouse variable region that includes unrearranged endogenous mousevariable region gene segments, unrearranged endogenous mouse D regiongene segments, and unrearranged endogenous mouse J region gene segments,wherein the mouse comprises a genetic modification of a mouse heavychain constant region as described herein. The mouse would thus becapable of making a mouse heavy chain antibody. Upon exposure to animmunogen of interest, the mouse would be capable of generating a heavychain antibody in accordance with the invention that is affinity maturedand capable of specifically binding an epitope of the immunogen ofinterest.

The genetically modified animal can be a mouse comprising a humantransgene that comprises unrearranged human variable region genesegments, unrearranged human D gene segments, and unrearranged human Jgene segments, a mu gene, and a sequence that allows fortrans-switching. The mouse would further comprise a mouse heavy chainconstant region modification as described herein. The mouse would bethus capable of making a fully human IgM antibody, and throughtrans-switching a chimeric human variable/mouse constant antibody,wherein the constant domain comprises a genetic modification asdescribed herein. Upon exposure to an immunogen of interest, the mousewould be capable of generating a heavy chain antibody in accordance withthe invention that is affinity matured and capable of specificallybinding an epitope of the immunogen of interest.

In Vitro Expression of Heavy Chain Antibodies

The inventors have established that a normal human or mouse heavy chainvariable region (hVR or mVR) can be expressed in an in vitro system inthe context of an IgG that lacks a functional CH1 domain. The inventorsexpressed an hVR from an unrearranged hVR minilocus in a mouse with awild-type mouse IgM. The expressed hVR was cloned onto an IgG2b lackinga CH1 domain, and the resulting hVR-IgG2ΔCH1 expressed and was secretedby a CHO cell transiently transfected with the hVR-IgG2ΔCH1 construct,effectively establishing that an hVR selected in a mouse having awild-type IgM can be expressed and secreted by a cell when switched toan IgG lacking a functional CH1 domain, i.e., as a heavy chain antibody.

The inventors constructed an in vitro system to express heavy chainsthat lack CH1 domains and that have hVRs or human camelized VRs (hVR*s)in CHO cells. The VRs were obtained from a RAG mouse that contained areplacement of the endogenous mouse heavy chain locus with a human heavychain variable region minilocus (having three human V region genesegments, 6-1, 1-2, and 1-3, all human DH gene segments, and all humanJH gene segments). The endogenous mouse immunoglobulin kappa and lambdalight chain loci were intact and functional.

Chimeric heavy chain (hVR-mFc) and camelized heavy chain (hVR*-mFc)constructs were made, for expression in CHO cells, using the VRsequences obtained from the mouse bearing the minilocus described above.The chimeric heavy chains were the product of normal V-D-J recombinationduring B cell development in the mouse to form a functional antibodycomprising a chimeric heavy chain (hVR-mFc) and a mouse light chain.hVR-mFc and hVR*-mFc constructs were made both having a CH1 domain andlacking a CH1 domain.

Transient transfection of hVR-mFc and hcVR-mFc constructs in CHO cellsshowed that in the absence of a CH1 domain, heavy chains having hVRs andhVR*s were expressed and remained soluble in the supernatant. In thepresence of a CH1 domain, heavy chains containing either hVRs or hVR*sdid not express in supernatant. This observation suggested that suchheavy chain antibodies could be made without employing camelid VHHdomains, e.g., with human or mouse VH domains, in heavy chain antibodiesthat lacked a CH1 domain.

Humanized Heavy Chain Antibodies

To produce a humanized version of a heavy chain antibody of the presentinvention, an animal homozygous for the modification is immunized withan antigen and once a specific immune response of the animal has beenestablished, cells from the spleen of the immunized animal are fusedwith a suitable immortal cell (e.g., a myeloma cell) to producehybridoma cells. Alternatively, the antibodies can be obtained directlyfrom B cells of the immunized animal. Supernatants from the hybridomacells (or, e.g., from isolated B cells) are screened for the presence ofantibody by enzyme-linked immunosorbent assay (ELISA) and antibodiesspecific for the antigen can be selected based on desiredcharacteristics.

Heavy chain variable region (VH) nucleic acids can be isolated fromhybridoma and/or B cells using standard molecular biology techniquesknown in the art (Sambrook, et al. 1989. Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor, N.Y.; Ausubel, et al. 1995.Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons). Once theVH nucleic acid sequence has been determined, the deduced amino acidsequence can be obtained and compared to other human VH sequences toidentify a group of related VH sequences that have a similar sequence.Related VH sequences can be obtained using antibody databases availableto those of skill in the art, e.g., The International ImMunoGeneTicsInformation System® (IMGT®). This comparison may be performed byalignment of the sequences accomplished either by eye or, alternatively,electronically by employing an alignment program (e.g., CLUSTAL). Inthis comparison, the complementary determining regions (CDRs) andframework regions (FRs) are identified. CDR and FR residues aredetermined according to a standard sequence definition (e.g., Kabat etal. 1987, Sequences of Proteins of Immunological Interest, NationalInstitutes of Health, Bethesda Md.; Chothia and Lesk, 1987. J. Mol Biol.196:901-917). Those skilled in the art will appreciate that there mayoccasionally exist discrepancies in methods of numbering and determiningthe CDR and FR regions of an immunoglobulin heavy chain sequence. Insuch cases, the structural definition is preferred, however, theresidues identified by the sequence definition method are consideredimportant FR residues for determination of which framework residues tosubstitute based on a comparison of heavy chain sequences.

Once aligned, substitutable positions in the VH sequences areidentified. If the identity of an amino acid at a position in theisolated VH sequence varies when compared to the other human VHsequences, that position is evaluated for the suitability of asubstitution at that position of the isolated VH sequence. Therefore,any position in the isolated VH sequence that varies with the otherrelated human VH sequence(s) to which it is being compared canpotentially serve as a position that could be substituted with the aminoacid at the corresponding position found in one or any of the otherrelated human VH sequences. Positions that share identity with the otherrelated human VH sequences, i.e., those that do not demonstratevariability, are determined to be nonsubstitutable positions. In variousembodiments, the above methods are employed to provide a consensus humanheavy chain antibody sequence.

A humanized heavy chain antibody for the purposes described herein is animmunoglobulin heavy chain amino acid sequence variant or fragmentthereof that is capable of binding to a predetermined antigen and thatcomprises a FR region having a substantially similar or an identicalamino acid sequence as compared with a human FR amino acid sequence, anda CDR having a substantially similar or an identical amino acid sequenceto a non-human CDR amino acid sequence. In general, a humanized heavychain antibody has one or more amino acid residues that are derived froma non-human source. Such residues are typically derived from a heavychain variable domain. Further, these residues may have associatedcharacteristics such as, for example, affinity and/or specificity aswell as other desirable biological activity associated with antibodyfunction.

In various embodiments, the humanized heavy chain antibody comprisessubstantially all of at least one, and in other embodiments at leasttwo, VH domains in which all or substantially all of the CDR regionscorrespond to those of a non-human VH domain and all or substantiallyall of the FR regions are those of a human VH domain sequence. Thehumanized heavy chain antibody will comprise a unique immunoglobulinconstant region (Fc), that in one embodiment lacks at least the CH1domain, and in one embodiment also lacks the hinge region of a human Fc.In one embodiment, the heavy chain antibody will not comprise a lightchain and will comprise the CH2 and CH3 regions of an immunoglobulin G(IgG) heavy chain constant region. In one embodiment, the constantregion of the heavy chain antibody will include the hinge, CH2 and CH3regions of the IgG heavy chain Fc. In one embodiment, the constantregion of the heavy chain antibody will include a CH1 region of an IgM.

The humanized heavy chain antibody will be selected from any class ofIgGs, including IgG1, IgG2, IgG3 and IgG4. In various embodiments, theconstant region may comprise sequences from more than one class of IgG,and selecting particular constant regions to optimize desired effectorfunctions is within the ordinary skill in the art.

In general, the heavy chain FR and heavy chain CDR regions of thehumanized heavy chain antibody need not correspond precisely to theparental sequences, e.g., the non-human heavy chain CDR or the humanheavy chain FRs may be altered by substitution, insertion or deletion ofat least one residue so that the heavy chain CDR or heavy chain FRresidue at a given site does not correspond to either the human heavychain FR sequence or the non-human heavy chain CDR sequence. Suchmutations, however, will not be extensive. In one embodiment, at least75% of the humanized heavy chain antibody residues will correspond tothose of the parental heavy chain FR and heavy chain CDR sequences, inanother embodiment 90%, and in another embodiment greater than 95%.

Humanized heavy chain antibodies as disclosed herein are, in oneembodiment, prepared by a process of analyzing parental sequences andvarious conceptual humanized composite sequences in silico, usingcomputer programs available and known to those skilled in the art.Sequence modifications to make humanized versions and/or for changingcharacteristics such as immunogenicity, affinity, etc. are madeemploying methods known in the art (e.g., U.S. Pat. No. 5,565,332Hoogenboom et al.; U.S. Pat. No. 5,639,641 Pedersen et al.; U.S. Pat.No. 5,766,886 Studnicka et al.; U.S. Pat. No. 5,859,205 Adair et al.;U.S. Pat. No. 6,054,297 Carter et al.; U.S. Pat. No. 6,407,213 Carter etal.; U.S. Pat. No. 6,639,055 Carter et al.; U.S. Pat. No. 6,849,425 Huseet al.; U.S. Pat. No. 6,881,557 Foote; U.S. Pat. No. 7,098,006 Gorman etal.; U.S. Pat. No. 7,175,996 Watkins et al.; U.S. Pat. No. 7,235,643Nicolaides et al.; U.S. Pat. No. 7,393,648 Rother et al.; U.S. Pat. No.7,462,697 Couto et al.).

In various embodiments, desired substitutions to a parental heavy chainantibody sequence to make a variant of a parental heavy chain antibodyare those that in one embodiment maintain, or in another embodimentincrease, the antigen binding activity of the parental heavy chainantibody. In general, a heavy chain antibody variant of a parental heavychain antibody has an antigen binding affinity that is at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90% or at least 100% (e.g., at least150%, at least 200%, at least 500%, at least 1000%, or up to at least10,000%) of the binding affinity of the parental heavy chain antibody toa particular antigen. In some embodiments, a variant heavy chainantibody will comprise a single substitution as compared to a parentalheavy chain antibody. However, in other embodiments, several aminoacids, e.g., up to about 5 or 10 or more, are substituted as compared tothe parental heavy chain antibody sequence that are derived from otherhuman heavy chain sequences that share identity at a given position.Substitutions in one embodiment are conservative (i.e., an amino acidsharing similar properties to the residue to be replaced), and inanother embodiment non-conservative (i.e., an amino acid sharingdifferent properties to the residue to be replaced). In variousembodiments, the resultant variant heavy chain antibody is tested toconfirm that the desired binding affinity and/or specificity has notbeen significantly decreased by the replacement residues. In someembodiments, an improved variant heavy chain antibody is produced by thesubstitution of amino acids from a different human heavy chain sequence.

Naturally occurring heavy chain antibodies (e.g., found in camelids)have been demonstrated to contain unique amino acid residues atpositions corresponding to the interface between heavy and light chainvariable regions in traditional antibody molecules (i.e., two heavychains and two light chains). These interface residues are known toaffect the proximity or orientation of the two chains relative to oneanother in traditional antibodies. Although these natural heavy chainantibodies are known to contain replacement of residues that correlatewith the absence of light chain variable regions, they retain theresidues at other positions in the sequence as compared to traditionalantibodies for preserving the characteristic immunoglobulin fold. Thesubstitutions found in natural heavy chain antibodies are L11S, V37F,G44E, L45R or L45C, W47G and additional cysteine residues thatcontribute to a disulfide bond between the CDR1 and CDR3 of the heavychain variable region. In some embodiments, heavy chain antibodies ofthe present invention may retain the residue of the parental antibody atthese positions. In other embodiments, the parental antibody may displaymutations at these positions that are associated with the residues innatural heavy chain antibodies. In some embodiments, it may be desirableto retain the same residue as is found in the parental heavy chainantibody at at least one of these positions or, in one embodiment, allof these positions when making a humanized heavy chain antibody derivedfrom an isolated VH sequence from a genetically modified mouse asdescribed herein. In various embodiments, a person of skill in the artwill understand that these interface residues are not reasonablyexpected to be involved in interchain interactions in heavy chainantibodies made by the genetically modified mouse as described herein.

Making Genetically Modified Animals

Genetic modifications for making an animal that expresses a heavy chainantibody are conveniently described by using the mouse as anillustration. A genetically modified mouse according to the inventioncan be made in a variety of ways, particular embodiments of which arediscussed below.

A schematic illustration (not to scale) of an IgG1 locus is provided inFIG. 1 (top) to show CH domain arrangement at the IgG1 locus. Asillustrated, domains CH1, CH2, and CH3 and the hinge region are presentin readily identifiable spans of nucleotide downstream of a switchregion.

A genetically modified mouse lacking a nucleotide sequence encoding aCH1 domain of an IgG1 but containing a hinge region can be made by anymethod known in the art. For example, a targeting vector can be madethat replaces the IgG1 gene with a truncated IgG1 lacking a CH1 domainbut containing the hinge. FIG. 2 illustrates a mouse genome (top)targeted by a targeting construct having a 5′ (with respect to thedirection of transcription of the genomic IgG1 gene) homology armcontaining sequence upstream of the endogenous CH1 domain, followed bynucleotide sequences that encode an IgG1 hinge, an IgG1 CH2 domain, anIgG1 CH3 domain, a drug selection cassette (e.g., a loxed resistancegene), and an IgG1 transmembrane domain, and a 3′ homology armcontaining sequence 3′ with respect to the transmembrane domain. Uponhomologous recombination at the locus and removal of the drug selectioncassette (e.g., by Cre treatment), the endogenous IgG1 is replaced by anIgG1 that lacks a CH1 domain (bottom of FIG. 2; lox site not shown).FIG. 1 (IgG1ΔCH1, middle) shows the structure of the resulting locus,which will express an IgG1 having a J region sequence fused to the hingesequence.

A genetically modified mouse lacking a nucleotide sequence encoding aCH1 domain of an IgG1 and lacking a nucleotide sequence encoding a hingeregion can be made by any method known in the art. For example, atargeting vector can be made that replaces the IgG1 gene with atruncated IgG1 lacking a sequence encoding a CH1 domain and lacking asequence encoding the hinge region. FIG. 3 illustrates a mouse genome(top) targeted by a targeting construct having a 5′ (with respect to thedirection of transcription of the genomic IgG1 gene) homology armcontaining sequence upstream of the endogenous CH1 domain, followed bynucleotide sequences that encode an IgG1 CH2 domain, an IgG1 CH3 domain,a drug selection cassette (e.g., a loxed resistance gene), and an IgG1transmembrane domain, and a 3′ homology arm containing sequence 3′ withrespect to the transmembrane domain. Upon homologous recombination atthe locus and removal of the drug selection cassette (e.g., by Cretreatment), the endogenous IgG1 gene is replaced by an IgG1 gene thatlacks a sequence encoding a CH1 domain (bottom of FIG. 3; lox site notshown). FIG. 1 (IgG1ΔCH1-Δhinge, bottom) shows the structure of theresulting locus, which will express an IgG1 having a J region sequencefused to the CH2 domain.

A genetically modified mouse lacking an IgG1 CH1 sequence (IgG1ΔCH1), orlacking an IgG1 CH1 sequence and lacking a hinge (IgG1ΔCH1-Δhinge), canbe further modified to favor usage of the modified IgG1 isotype bydeleting one or more other IgG isotypes, e.g., by deleting orfunctionally disabling sequences encoding IgG2b and IgG2a. For example,a targeting construct is made having a 5′ homology arm containingsequence upstream of the endogenous hinge region sequence (or upstreamof the endogenous CH1 domain sequence), sequences that encode the IgG1CH2 and CH3 domains, a drug selection cassette followed by a sequenceencoding the IgG1 transmembrane domain, followed by another drugselection cassette if desired. Upon homologous recombination at thelocus and removal of the drug selection cassette(s) (e.g., by Cretreatment), the endogenous heavy chain constant locus contains only twoIgG genes: an endogenous IgG3 and the IgG1ΔCH1 (see FIG. 4, bottom;recombinase site(s) not shown; see FIG. 6, bottom) or IgG1ΔCH1-Δhinge(see FIG. 5, bottom; recombinase site(s) not shown; see FIG. 6, bottom).

An IgG1 expressed in a genetically modified mouse having anIgG1ΔCH1-Δhinge or an IgG1ΔCH1ΔIgG2aΔIgG2b allele will have a structureas shown on the right panel of FIG. 10, i.e., the VH domain will befused to the CH2 domain. The left panel of FIG. 10 provides, forcomparison, a wild-type IgG1 antibody, showing its CH1 domain linked viaa hinge region to the CH2 domain, and linked by disulfide linkage to thelight chain constant domain CL. In contrast, the antibody made by thegenetically modified mouse lacks the hinge and CH1 domains and thuslacks any CL domain.

Genetically modified mice as described above, and others, are made byintroducing a suitable targeting construct into a suitable mouse ES cell(in one or more independent targetings), and positive clones comprisinga marker or selection cassette of the targeting construct are identifiedand grown. Clones are then employed as donor ES cells in a host embryounder conditions suitable for making a chimeric mouse or a fully EScell-derived mouse. The marker or selection cassette can be optionallyremoved, either at the ES cell stage or in the chimeric or EScell-derived mouse, e.g., by employing a loxed cassette and breeding toa Cre-containing strain, or by electroporating the ES cell with a Creexpression vector.

A genetically modified mouse having an IgG1ΔCH1-Δhinge allele(heterozygous) was made in accordance with an embodiment of theinvention. Serum was isolated from the mouse and blotted in a Western(reducing conditions) using an anti-mouse IgG1 antibody to detect heavychain. In contrast to a wild-type mouse, which displayed a bandcorresponding in size to a wild-type IgG1 heavy chain, the mousegenetically modified to contain the IgG1ΔCH1-Δhinge allele alsoexpressed a heavy chain that reacted with anti-mouse IgG1 antibody thathad the expected size of a heavy chain antibody consisting of the VH,CH2, and CH3 domains (see FIG. 8).

EXAMPLES Example 1 In vitro Expression of Heavy Chain Antibodies

Chimeric heavy chain constructs were made using molecular biologytechniques (e.g., see Maniatis et al. 1982. Molecular Cloning: ALaboratory Manual. Cold Spring Harbor Laboratory) to fuse human variableregions with a murine IgG2b (mIgG2b) constant region. The human variablegene segment for each construct was a full-length human variable genesegment containing both exons (i.e., leader sequence plus maturesequence), identified from an hVR of an IgM isolated from a naive RAGmouse that contained a replacement of the endogenous mouseimmunoglobulin heavy chain locus with three hVR gene segments, all hDHgene segments, and all hJH gene segments. The light chain of the IgMantibody was a mouse light chain.

Two versions of the mIgG2b sequence were used; one with and one withouta CH1 domain. Several other constructs were also made to serve astransfection and expression controls. A first control construct was madeusing a cytokine receptor fused to the CH2 and CH3 domains of mouseIgG2a (mIgG2a) constant region (Control I). Two other controls wereconstructed by fusing a murine ROR signal sequence to a murine IgG2asequence with and without CH1 domains (Control II and III,respectively).

Camelized versions of each human variable region were also made usingPCR site-directed mutagenesis techniques (e.g., see Hutchinson et al.1978. Mutagenesis at a specific position in a DNA sequence. J. Biol.Chem. 253(18):6551-60). Two specific primer sets were used for eachvariable region to create specific mutations within the human variableregion sequence resulting in a human variable region sequence containingcamel-like features. Primers L1 (SEQ ID NO:1) and HH1.2 mut BOT (SEQ IDNO:2) were used to amplify one product comprising the 5′ half of thevariable region while primers HH1.2 mut TOP (SEQ ID NO:3) and m18.3.1(SEQ ID NO:4) were used to amplify the 3′ half of the variable region.These products were purified and mixed together to serve as a templatefor a third PCR reaction using primers L1 and m18.3.1. The resultingcamelized human variable region PCR product was cloned, purified andconfirmed by sequencing.

The full length heavy chain constructs (variable and constant) were madeby amplifying the human variable regions (camelized and non-camelized)and constant regions with primers containing restriction enzyme sites toallow for subsequent ligation together via cohesive ends. All fulllength heavy chain constructs were cloned into expression vectors,purified and confirmed again by sequencing. Table 1 sets forth eachheavy chain construct, their SEQ ID NOs and a short description for eachconstruct.

TABLE 1 SEQ ID NO Construct Description (DNA/Protein) hVR-mFcNon-camelized human variable region fused 5/6 to mouse IgG2b hVR*-mFcCamelized human variable region fused to 7/8 mouse IgG2b hVR-Non-camelized human variable region fused  9/10 mFcΔCH1 to mouse IgG2blacking a CH1 domain hVR*- Camelized human variable region fused to11/12 mFcΔCH1 mouse IgG2b lacking a CH1 domain

Chimeric heavy chain constructs were transiently transfected intoChinese Hamster Ovary cells (CHO-K1) to analyze expression in theabsence of immunoglobulin light chain. Supernatants and cell lysateswere examined by Western blot to detect presence of heavy chain usinghorseradish peroxidase (HRP) conjugated anti-mouse IgG antibody(Promega) by chemilumescence. All the chimeric heavy chain constructswere transiently transfected six (6) independent times. A representativeWestern blot of the transfections is shown in FIG. 7.

All chimeric heavy chain constructs, with and without the CH1 domain, aswell as the control constructs, were detected in the cell lysate. Onlyconstructs lacking a CH1 domain were observed in the supernatants (FIG.7, left). Control I and Control III (mouse Fc protein lacking a CH1domain) were also detected (FIG. 7), but mouse Fc protein containing aCH1 domain was not detected. Both non-camelized and camelized heavychain constructs containing a CH1 domain were not detected in thesupernatant for any transfection (FIG. 7, right). However, bothnon-camelized and camelized human heavy chain constructs lacking a CH1domain were detected in the supernatant for all transfections. Together,the results establish that hVRs (normal or camelized) that lack a CH1domain can be expressed and secreted from transiently transfected CHOcells in the absence of immunoglobulin light chain, whereas hVRs (normalor camelized) that contain a CH1 domain could not be secreted in theabsence of light chain.

Example 2 Modification of the Mouse Heavy Chain IgG1 Constant Region

A. Preparation of a Mouse IgG1-CH1-Hinge Targeting Vector (FIG. 3)

A targeting construct for introducing a deletion of the CH1 and hingeregions of the mouse IgG1 constant domain for the C57BL/6 allele from anES cell of a VELOCIMMUNE® mouse (described below) was constructed.

The targeting construct was made using VELOCIGENE® technology (see,e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al. (2003)High-throughput engineering of the mouse genome coupled withhigh-resolution expression analysis, Nature Biotech. 21(6):652-659) tomodify the Bacterial Artificial Chromosome (BAC) BMQ 70p08. BMQ 70p08BAC DNA was modified to delete the CH1 and hinge regions of the IgG1constant domain while leaving the remainder of the IgG1 gene intact(e.g., CH2, CH3 and transmembrane exons).

Briefly, upstream and downstream homology arms were made employingprimers m102 (SEQ ID NO:13) and m104 (SEQ ID NO:14) and m100 (SEQ IDNO:15) and m99 (SEQ ID NO:16), respectively. These homology arms wereused to make a cassette that deleted the CH1 and hinge regions of theIgG1 constant domain while retaining the CH2, CH3 and transmembraneregions of the IgG1 constant domain (see, e.g., FIG. 3). The targetingconstruct included a loxed hygromycin resistance gene positioned betweenthe CH3 and transmembrane domain exons of the IgG1 gene. Genes upstreamof the CH1 and hinge exons (e.g., IgG3, IgD, IgM) and downstream of theIgG1 transmembrane exon (e.g., IgG2b, IgG21, IgE, IgA, etc.) wereunmodified by the targeting construct. Switch regions for all constantdomains were unmodified by the targeting construct. The nucleotidesequence across the deletion included the following, which indicates asplice acceptor sequence that is present at the deletion point:TGACAGTGTA ATCACATATA CTTTTTCTTG T(AG)TCCCAGA AGTATCATC (SEQ ID NO:17).The deletion sequence comprises a splice acceptor (the AG containedwithin parentheses above) with pre-CH1 sequences 5′ of the spliceacceptor and CH2 exon sequences 3′ of the splice acceptor.

B. Preparation of a Mouse IgG1-CH1 Targeting Vector (FIG. 2)

A second targeting construct for introducing a deletion of the CH1 ofthe mouse IgG1 constant domain for the 129/SvEvTac allele from an EScell of a VELOCIMMUNE® mouse (described below) was constructed in asimilar fashion as described in section A of this Example.

The targeting construct was made using VELOCIGENE® technology (see,e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al. (2003)High-throughput engineering of the mouse genome coupled withhigh-resolution expression analysis, Nature Biotech. 21(6):652-659) tomodify the Bacterial Artificial Chromosome (BAC) BMQ 70p08. BMQ 70p08BAC DNA was modified to delete the CH1 region of the IgG1 constantdomain while leaving the remainder of the IgG1 gene intact (e.g., hinge,CH2, CH3 and transmembrane exons; see FIG. 2).

The homology arms for the second targeting construct were the same asthose for the CH1-Hinge targeting vector (as described above in sectionA of this Example). These homology arms were used to make a cassettethat deleted the CH1 region of the IgG1 constant domain while retainingthe hinge, CH2, CH3 and transmembrane regions of the IgG1 constantdomain (see, e.g., FIG. 2). The targeting construct included a loxedhygromycin resistance gene positioned between the CH3 and transmembranedomain exons of the IgG1 gene. Genes upstream of the CH1 exon (e.g.,IgG3, IgD, IgM) and downstream of the IgG1 transmembrane exon (e.g.,IgG2b, IgG21, IgE, IgA, etc.) were unmodified by the targetingconstruct. Switch regions for all constant domains were unmodified bythe targeting construct. The nucleotide sequence across the deletionincluded the following, which indicates a splice acceptor sequence thatis present at the deletion point: TGACAGTGTA ATCACATATA CTTTTTCTTGT(AG)TGCCCAG GGATTGTGGT TGTAAGCCTT GCATATGTAC AGGTAAGTCA GTAGGCCTTTCACCCTGACC C (SEQ ID NO:64). The deletion sequence comprises a spliceacceptor (the AG contained within parentheses above) with pre-CH1sequences 5′ of the splice acceptor and hinge exon sequences 3′ of thesplice acceptor.

Example 3 Modification of the Mouse Heavy Chain Constant Region in ESCells

A. Targeting Mouse ES Cells with an IgG1-CH1-Hinge Targeting Vector

A mouse ES cell was targeted with the targeting construct describedabove (i.e., a targeting construct introducing a deletion of the CH1 andhinge regions of the IgG1 gene). The ES cell was from a VELOCIMMUNE®mouse that was a 50/50 mix of a 129 strain and a C57BL/6 strain, bearinggenetic modifications that comprise replacement of mouse heavy and lightchain variable region gene segments with unrearranged human heavy andlight chain variable region gene segments. The 129 strain employed tocross with C57BL/6 is a strain that comprises a replacement of mouseheavy chain and light chain variable region gene segments with humanheavy chain and light chain variable region gene segments.

The heterozygous VELOCIMMUNE® mice bear a single set of endogenous mouseheavy chain constant region genes from the 129 strain at one allele anda single set of endogenous mouse heavy chain constant region genes fromthe C57BL/6 strain at the other allele. The 129 heavy chain allele iscontiguous with a locus of heavy chain variable region gene segmentsthat are human heavy chain variable region gene segments that havereplaced the endogenous mouse heavy chain variable region gene segments(i.e., at the endogenous mouse locus). The BL/6 heavy chain allele iscontiguous with wild-type mouse heavy chain variable region genesegments. The VELOCIMMUNE® mice also bear wild-type endogenous mouselight chain constant region genes. Thus, by targeting the 129 allelewith a construct comprising an IgG, D, E, or A CH1 deletion a chimerichuman/mouse heavy chain antibody could be produced, whereas by targetingthe C57BL/6 allele with a similar construction, a fully mouse heavychain antibody lacking a CH1 domain and lacking a hinge could beproduced.

ES cells from the VELOCIMMUNE® mice described above were electroporatedwith linearized targeting vector of section A in Example 2 and selectedfor the presence of the hygromycin resistance gene.

B. Targeting Mouse ES Cells with an IgG1-CH1 Targeting Vector

In a similar fashion, a mouse ES cell was targeted with the CH1targeting construct described in section B of Example 2 (see also FIG.2). The ES cell was from a VELOCIMMUNE® mouse that was a 50/50 mix of a129/SvEvTac strain and a C57BL/6 strain, bearing genetic modificationsthat comprise replacement of mouse heavy and light chain variable regiongene segments with unrearranged human heavy and light chain variableregion gene segments. The 129/SvEvTac strain employed to cross withC57BL/6 is a strain that comprises a replacement of mouse heavy chainand light chain variable region gene segments with human heavy chain andlight chain variable region gene segments.

The heterozygous VELOCIMMUNE® mice bear a single set of endogenous mouseheavy chain constant region genes from the 129/SvEvTac strain at oneallele and a single set of endogenous mouse heavy chain constant regiongenes from the C57BL/6 strain at the other allele. The 129/SvEvTac heavychain allele is contiguous with a locus of heavy chain variable regiongene segments that are human heavy chain variable region gene segmentsthat have replaced the endogenous mouse heavy chain variable region genesegments (i.e., at the endogenous mouse locus). The BL/6 heavy chainallele is contiguous with wild-type mouse heavy chain variable regiongene segments. The VELOCIMMUNE® mice also bear wild-type endogenousmouse light chain constant region genes. Thus, by targeting the129/SvEvTac allele with a construct comprising an IgG, D, E, or A CH1deletion a chimeric human/mouse heavy chain antibody could be produced,whereas by targeting the C57BL/6 allele with a similar construction, afully mouse heavy chain antibody lacking a CH1 domain and lacking ahinge could be produced.

ES cells from the VELOCIMMUNE® mice described above were electroporatedwith linearized targeting vector, described in section B in Example 2,and selected for the presence of the hygromycin resistance gene.

Example 4 Generation of Mice Carrying a Modified IgG1 Constant Region

A. Mice Carrying an IgG1-CH1-Hinge Deletion

Targeted ES cells described above were used as donor ES cells andintroduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method(see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. (2007) F0generation mice that are essentially fully derived from the donorgene-targeted ES cells allowing immediate phenotypic analyses NatureBiotech. 25(1):91-99. VELOCIMICE® (F0 mice fully derived from the donorES cell) bearing targeted C57BL/6 IgG1 alleles were identified bygenotyping using a modification of allele assay (Valenzuela et al.,supra) that detected the presence of sequences positioned upstream anddownstream of the deleted hinge and CH1 regions.

Mice genotyped for the IgG1 CH1 and hinge deletion (in the C57BL/6allele, i.e., the mouse allele) were bred to a Cre deleter mouse strain(see, e.g., International Patent Application Publication No. WO2009/114400) in order to remove the loxed hyg cassette downstream of theIgG1 CH3 exon and upstream of the IgG1 transmembrane exon, introduced bythe targeting construct (see, e.g., FIG. 3). Pups were genotyped and apup heterozygous for the IgG1 CH1 and hinge deletion was selected toexamine IgG1 heavy chain expressed from the C57BL/6 allele in the pup'sserum.

B. Mice Carrying an IgG1-CH1 Deletion

In a similar fashion, targeted ES cells carrying a deletion of the IgG1CH1 region were used as donor ES cells and introduced into an 8-cellstage mouse embryo by the VELOCIMOUSE® method (see, e.g., U.S. Pat. No.7,294,754 and Poueymirou et al. (2007) F0 generation mice that areessentially fully derived from the donor gene-targeted ES cells allowingimmediate phenotypic analyses Nature Biotech. 25(1):91-99. VELOCIMICE®(F0 mice fully derived from the donor ES cell) bearing targeted129SvEv/Tac alleles were identified by genotyping using a modificationof allele assay (Valenzuela et al., supra) that detected the presence ofsequences positioned upstream and downstream of the deleted CH1 region.

Mice genotyped for the IgG1 CH1 deletion (in the 129/SvEvTac allele,i.e., the human allele) were bred to a Cre deleter mouse strain (see,e.g., International Patent Application Publication No. WO 2009/114400)in order to remove the loxed hyg cassette downstream of the IgG1 CH3exon and upstream of the IgG1 transmembrane exon, introduced by thetargeting construct (see, e.g., FIG. 2). Pups were genotyped and a puphomozygous for the IgG1 CH1 deletion was selected to examine modifiedIgG1 heavy chain expression.

Example 5 Heavy Chain Antibodies from Mice Carrying a Modified IgG1 Gene

A. IgG1-ΔCH1-ΔHinge Mice

A mouse pup identified above as containing the CH1 and hinge deletion,and a wild-type pup, were bled and sera from the bled mice were preparedfor Western blotting to identify any expressed IgG in the sera using ananti-mIgG1 antibody. Briefly, 10 μL of a 1:100 dilution of mouse serawas used in reducing SDS-PAGE, and the gel was transferred to a PVDFmembrane. The blot was blocked overnight with 5% nonfat milk inTris-Buffered Saline with 0.05% Tween-20 (TBST; Sigma), washed 4 timesfor 5 minutes per wash with TBST, and then exposed to primary antibody(goat anti-mIgG1 conjugated to HRP, Southern Biotech) diluted 1:1,000 in1% nonfat milk in TBST for two hours at room temperature. The blot waswashed 6 times for 5 minutes per wash. The blot was developed for 5minutes with SUPERSIGNAL™ West Pico Chemiluminescent Substrate (ThermoScientific) and then exposed to film for 1 minute.

Serum from the VELOCIMOUSE® (50% wild-type BL/6; 50% ΔCH1-Δhinge BL/6)derived from the targeted donor ES cell revealed a mixture of bands: oneband of about 57.5 kD, the expected size for a wild-type IgG, and oneband at about 45 kD, the expected size for an IgG lacking a CH1 domainand a hinge (FIG. 8). The results are consistent with the VELOCIMOUSE®expressing a normal mouse heavy chain from the wild-type BL/6 allele anda ΔCH1/Δhinge mouse heavy chain from its ΔCH1-Δhinge BL/6 allele. Thisresult establishes that genetically modified mice bearing a functionalIgM gene and an IgG gene that lacks a CH1 domain and a hinge domain arecapable of expressing heavy chain antibodies in serum.

B. IgG1-ΔCH1 Mice

In a similar fashion, mouse pups homozygous for the CH1 deletion, andwild-type pups, were bled. Plasma and serum (for five homozygotes; twowild-type) from the bled mice were prepared for Western blotting toidentify any expressed IgG in the sera using an anti-mIgG1 antibody(described above). Western blots of serum and plasma from micehomozygous for the IgG1-ΔCH1 deletion revealed a mixture of bands: oneband of about 45 kD, the expected size for a single chain IgG1 lacking aCH1 domain, and one band at about 75 kD, the expected size for a dimerIgG lacking a CH1 domain (data not shown). The results are consistentwith the homozygous VELOCIMICE® expressing an IgG1-ΔCH1 heavy chain fromeither one or both heavy chain loci. This result establishes thatgenetically modified mice bearing a functional IgM gene and an IgG genethat lacks a CH1 domain are capable of expressing heavy chain antibodiesin the peripheral lymphocyte compartment of the animals' immune system.

Example 6 Characterization of Mice Homozygous for IgG1-CH1-HingeDeletion

VELOCIMICE® heterozygous for the CH1-hinge deletion were bred togetherto obtain mice homozygous for the deletion. Four mouse pups wereidentified as homozygous for IgG1 ΔCH1-Δhinge. These four mice and awild-type mouse were bled and sera from the bled mice were prepared forWestern blotting to identify any expressed IgG in the sera using ananti-mIgG1 antibody (as described above). FIG. 9 shows the filmdeveloped from the PVDF-membrane used in this experiment. Serum wasdiluted 1:5 and 1:10 and 10 μL of each dilution was loaded onto the gelside-by-side for each mouse. On the top portion of the gel images, thelanes are labeled for each mouse as well as IgG1 (1) and IgG2a (2a)controls.

Serum from the wild-type mouse showed an expected pattern for awild-type mouse that expresses normal antibodies comprising two heavychains and two light chains (approximately 150 kD). All four mice(homozygous for IgG1 ΔCH1-Δhinge) each showed a mixture of bands: oneband of about 150 kD, the expected size for a wild-type IgG other thanIgG1 (e.g., IgG2a, IgG2b or IgG3), and one band at about 45 kD, theexpected size for an IgG lacking a CH1 domain and a hinge (FIG. 9).These results are consistent with the mice expressing an IgG1 heavychain antibody lacking a CH1 domain and a hinge region and lacking alight chain. This result further establishes that genetically modifiedmice bearing a functional IgM gene and an IgG gene that lacks a CH1domain and a hinge region are capable of expressing heavy chainantibodies in serum.

In another experiment, serum expression of IgG was determined from micehomozygous for the IgG1 ΔCH1-Δhinge using an ELISA assay. Briefly,antibodies specific for either mIgG1 or mlgG2b (Pharmingen) wereseparately diluted and 100 μl/well was coated onto plates at 2 μg/mL in1×PBS (Irvine Scientific) and incubated at 4° C. overnight. Thefollowing day the plates were washed four times with PBS with 0.05%Tween-20 (PBST; Sigma). After the fourth wash, plates were blocked with250 μL/well of PBST with 5% BSA (Sigma) and incubated at roomtemperature for one hour. Serum and standards were serially diluted(dilution factor of 0.316) in PBST in 0.5% BSA down the plate (from topto bottom) at a starting concentration of 400 ng/mL (mIgG1) or 600 ng/mL(mlgG2b). After blocking, the plates were washed again four times withPBST. Following the fourth wash, 100 μL of serum or standard was addedto the plates and incubated for one hour at room temperature. The plateswere again washed four times with PBST. Following the washes, 100 μL ofa biotinylated detection antibody (10 ng/mL of rat anti-mIgG1 or 250ng/mL of anti-migG2b; Pharmingen) was added to the plates and incubatedfor one hour at room temperature. The plates were again washed asdescribed above. Following the wash, 100 μL/well of a 1:20,000 dilutionof horseradish peroxidase conjugated to streptavidin (HRP-SA) in PBSTwas added to the plates and the plates were incubated for 30 minutes atroom temperature. The plates were then washed six times with PBST, afterwhich 100 μL/well of a 1:1 dilution of Substrate A and B (BD OPTEIA™; BDBiosciences) was added and the plates were maintained in the dark. Thereaction was developed in the dark and stopped as desired (approx. 15minutes) with 1N phosphoric acid. Stopped reactions were read on aWallac 1420 Work Station VICTOR™ Plate Reader at an absorptionwavelength of 450 nm (1.0 sec/reading) and the results plotted on graphs(FIG. 11).

Serum from wild-type mice showed normal levels of IgG1 and IgG2b. Micehomozygous for IgG1 ΔCH1-Δhinge were capable of expressing an IgG1lacking a CH1 domain and a hinge region in the periphery (serum; leftside of FIG. 11). Further, serum levels of other IgG isotypes (e.g.,IgG2b) were not noticeably reduced from wild-type levels (right side ofFIG. 11). This result further establishes that genetically modified micebearing a functional IgM gene and an IgG gene that lacks a CH1 domainand a hinge region are capable of expressing a modified IgG1 isotype(i.e., lacking a CH1 domain and a hinge) that can be detected in serum.

Example 7 Analysis of V-D-J Rearrangements in IgG1 Modified Mice

A. Mice Homozygous for an IgG1-CH1-Hinge Deletion

Mice homozygous for the IgG1 ΔCH1-Δhinge modification were analyzed forV-D-J recombination and heavy chain gene usage by reverse-transcriptasepolymerase chain reaction (RT-PCR) using RNA isolated from splenocytes.

Briefly, spleens were harvested and perfused with 10 mL RPMI-1640(Sigma) with 5% HI-FBS in sterile disposable bags. Each bag containing asingle spleen was then placed in a STOMACHER™ (Seward) and homogenizedat a medium setting for 30 seconds. Homogenized spleens were filteredusing a 0.7 μm cell strainer and then pelleted with a centrifuge (1000rpm for 10 minutes) and red blood cells (RBCS) were lysed in BD PHARMLYSE™ (BD Biosciences) for three minutes. Splenocytes were diluted withRPMI-1640 and centrifuged again followed by resuspension in 1 mL of PBS(Irvine Scientific). RNA was isolated from pelleted splenocytes usingstandard techniques known in the art.

RT-PCR was performed on splenocyte RNA using a set of degenerate primersspecific for mouse heavy chain variable region (VH) gene segments(Novagen) and a mouse IgG1 CH2 primer (CGATGGGGGC AGGGAAAGCT GCAC; SEQID NO:40). PCR products were gel-purified and cloned into pCR2.1-TOPO TA(Invitrogen) and sequenced with M13 Forward (GTAAAACGAC GGCCAG; SEQ IDNO:41) and M13 Reverse (CAGGAAACAG CTATGAC; SEQ ID NO:42) primerslocated within the vector sequence at positions flanking the cloningsite. Nineteen clones were sequenced to determine heavy chain gene usageand sequence of the junction of the rearranged VH and the CH2 of theIgG1 constant region (Table 2).

TABLE 2 Heavy Chain Gene Usage Clone V_(H) D_(H) J_(H) B1 1-58 3-2 2 B21-26 4-1 1 B3 1-50  2-14 2 B4 1-58 3-2 2 B5 14-2  4-1 4 D2 3-6  1-1 4 D514-1  3-3 2 D6 14-2  4-1 3 D7 3-6  1-1 4 E2 7-1  3-1 4 E3 1-50  2-14 2E4 1-50  2-14 2 E7 1-50  2-14 2 E8 1-72 1-1 4 E10 1-42 1-1 1 F6 5-6  1-11 F7 5-6  1-1 1 F8 5-6  1-1 1 F10 5-6  1-1 1

FIG. 12 shows the sequence alignment of the VH domains rearranged to theCH2 of the IgG1 constant region for eleven of the nineteen RT-PCRclones. The sequences shown in FIG. 12 illustrate unique rearrangementsinvolving different mouse heavy chain V, D and J gene segments and mouseIgG1 devoid of CH1 and hinge regions. Mice homozygous for a deletion ofthe CH1 and hinge regions of the endogenous IgG1 constant region genewere able to produce heavy chains containing mouse VH domains operablylinked to a CH2-CH3 region from a mouse IgG1 constant region devoid ofCH1 and hinge regions and produce B cells that expressed mouse IgG1heavy chains devoid of CH1 and hinge regions and lacking a light chain(FIGS. 8 and 9). These rearrangements demonstrate that the modified lociwere able to independently rearrange mouse heavy chain gene segments inmultiple, independent B cells in these mice to produce heavy chainantibodies that are similar to those normally found in camels. Further,this Example demonstrates that the deletion of the endogenous IgG1 CH1and hinge regions did not render the locus inoperable or preventrecombination involving the modified IgG1 constant region. These micemade functional heavy chain antibodies containing an IgG1 devoid of CH1and hinge regions as part of the endogenous repertoire without anydetectable defect in B cell development.

B. Mice Homozygous for an IgG1-CH1 Deletion

In a similar fashion, mice homozygous for the IgG1 ΔCH1 modificationwere analyzed for V-D-J recombination and human heavy chain gene usageby reverse-transcriptase polymerase chain reaction (RT-PCR) using RNAisolated from splenocytes.

Briefly, spleens were isolated from two homozygous IgG1-ΔCH1 mice asdescribed above in section A of this Example. CD19⁺ B cells wereisolated using magnetic cell sorting (MACS, Miltenyi Biotec) from pooledsplenocytes. RNA was extracted from the sorted CD19⁺ B cells usingQiagen ALLPREP™ DNA/RNA mini kit (Qiagen). First-strand cDNA wassynthesized with SUPERSCRIPT™ III Reverse Transcriptase and Oligo (dT)20primers (Invitrogen). The cDNA was then used as a template for PCRperformed with a 3′ mouse IgG1 hinge specific primer and 5′ degenerateprimers designed to bind human heavy variable leader sequences (Table3). PCR products were cloned into pCR2.1 TOPO™ TA vector (Invitrogen)and sequenced with M13 Forward and M13 Reverse primers (as describedabove in section A of this Example).

TABLE 3 SEQ ID Primer Sequence (5′-3′) NO: hVHL-1TCACCATGGA CTGSACCTGG A 43 hVHL-2 CCATGGACAC ACTTTGYTCC AC 44 hVHL-3TCACCATGGA GTTTGGGCTG AGC 45 hVHL-4 AGAACATGAA ACAYCTGTGG TTCTT 46hVHL-5 ATGGGGTCAA CCGCCATCCT 47 hVHL-6 ACAATGTCTG TCTCCTTCCT CAT 48 3′mlgG1 GCAAGGCTTA CAACCACAAT C 49 Hinge

To determine heavy chain gene usage in mice homozygous for IgG1 ΔCH1,twenty-eight RT-PCR clones were sequenced. Within these clones, sevenunique rearrangements of human V, D and J gene segments were observed(Table 4).

TABLE 4 Heavy Chain Gene Usage Clone V_(H) D_(H) J_(H) A2 1-69  6-19 6A5 1-69 6-7 4 A8 1-8  4-4 4 C2 1-18 6-6 2 C4 1-18  3-16 6 D9 1-18 6-6 4H8 1-18 1-7 4

FIG. 13 shows the sequence alignment of the VH domains rearranged to thehinge-CH2-CH3 of the IgG1 constant region for the seven rearrangementsshown in Table 4. The sequences shown in FIG. 13 illustrate uniquerearrangements involving different human heavy chain V, D and J genesegments and mouse IgG1 devoid of the CH1 region. Mice homozygous for adeletion of the CH1 region of the endogenous IgG1 constant region genewere able to produce heavy chains containing human VH domains operablylinked to a hinge-CH2-CH3 region from a mouse IgG1 constant regiondevoid of CH1 and produce B cells that expressed mouse IgG1 heavy chainsdevoid of CH1 regions and lacking a light chain (data not shown). Theserearrangements demonstrate that either one or both modified loci (IgG1ΔCH1-Δhinge and IgG1 ΔCH1) were able to independently rearrange heavychain gene segments (mouse and human) in multiple, independent B cellsin these mice to produce heavy chain antibodies that are similar tothose normally found in camels. Further, this Example demonstrates thatthe deletion of the endogenous IgG1 CH1 did not render the locusinoperable or prevent recombination involving human heavy chain V, D andJ gene segments and the modified mouse IgG1 constant region. These micemade functional heavy chain antibodies containing human heavy chain Vdomains and a mouse IgG1 devoid of CH1 as part of the endogenousrepertoire without any detectable defect in B cell development.

We claim:
 1. A genetically modified non-human animal whose germlinegenome comprises a modified endogenous non-human immunoglobulin (Ig)heavy chain locus, wherein the modified endogenous non-human Ig heavychain locus comprises unrearranged human Ig heavy chain variable regionV_(H), D_(H), and J_(H) gene segments operably linked to a modifiedendogenous non-human Ig heavy chain constant region nucleic acidsequence comprising (i) an endogenous non-human IgG1 constant regiongene modified to (1) comprise a nucleotide sequence comprising from 5′to 3′: an endogenous non-human switch region, an IgG1 CH2 exon, an IgG1CH2 intron, and an IgG1 CH3 exon, and a deletion in a sequence encodingan IgG1 CH1 domain and an IgG1 hinge region such that the nucleotidesequence further comprises, at the deletion point, a nucleotide sequenceset forth as SEQ ID NO:17, and (2) encode an endogenous non-human IgG1constant domain that is devoid of a CH1 domain and a hinge region andthat consists essentially of an endogenous non-human IgG1 CH2 domain andan endogenous non-human IgG1 CH3 domain; and (ii) an endogenousnon-human IgM constant region gene that encodes an endogenous non-humanIgM constant domain comprising an IgM CH1 domain, such that thenon-human animal expresses (i) a chimeric human/non-human IgG1immunoglobulin heavy chain that comprises, from N-terminal toC-terminal, a human Ig heavy chain variable domain operably linked tothe endogenous non-human IgG1 constant domain that is devoid of a CH1domain and a hinge region, and that consists essentially of anendogenous non-human IgG1 CH2 domain and an endogenous non-human IgG1CH3 domain, and (ii) a chimeric human/non-human IgM immunoglobulin heavychain that comprises a human Ig heavy chain variable domain operablylinked to the endogenous non-human IgM constant domain comprising an IgMCH1 domain; and wherein the non-human animal is a rat or a mouse.
 2. Thenon-human animal of claim 1, wherein the non-human animal expresses: (a)antibodies comprising an endogenous non-human IgG3 constant domaincomprising an IgG3 CH1 domain, an IgG3 hinge region, an IgG3 CH2 domain,and an IgG3 CH3 domain; (b) antibodies comprising an endogenousnon-human IgG2a constant domain comprising an IgG2a CH1 domain, an IgG2ahinge region, an IgG2a CH2 domain, and an IgG2a CH3 domain; and (c)antibodies comprising an endogenous non-human IgG2b constant domaincomprising an IgG2b CH1 domain, an IgG2b hinge region, an IgG2b CH2domain, and an IgG2b CH3 domain.
 3. The non-human animal of claim 2,further characterized in that the non-human animal additionallyexpresses: (d) antibodies comprising an endogenous non-human IgDconstant domain comprising an IgD CH1 domain, an IgD hinge region, anIgD CH2 domain, and an IgD CH3 domain; (e) antibodies comprising anendogenous non-human IgA constant domain comprising an IgA CH1 domain,an IgA hinge region, an IgA CH2 domain, and an IgA CH3 domain; and (g)antibodies comprising an endogenous non-human IgE constant domaincomprising an IgE CH1 domain, an IgE CH2 domain, and an IgE CH3 domain.4. The non-human animal of claim 1, wherein the non-human animalexpresses the chimeric human/non-human IgM immunoglobulin heavy chainassociated with a cognate light chain.
 5. The non-human animal of claim1, wherein the non-human animal is from a mouse strain selected from thegroup consisting of a 129 strain, a C57BL/6 strain, and a mixed129xC57BL/6 strain.
 6. The non-human animal of claim 5, wherein themouse is 50% 129 and 50% C57BL/6.
 7. The non-human animal of claim 1,wherein the unrearranged human Ig heavy chain variable region V_(H),D_(H), and J_(H) gene segments replace one or more endogenous non-humanIg heavy chain variable region gene segments.
 8. The non-human animal ofclaim 1, wherein the modified endogenous non-human Ig heavy chainconstant region nucleic acid sequence further comprises: (iii) adeletion of an endogenous non-human IgD constant region gene; (iv) adeletion of an endogenous non-human IgG3 constant region gene; (v) adeletion of an endogenous non-human IgG2b constant region gene; (vi) adeletion of an endogenous non-human IgE constant region gene; and (vii)a deletion of an endogenous non-human IgA constant region gene.
 9. Thenon-human animal of claim 1, wherein the modified endogenous non-humanIg heavy chain constant region nucleic acid sequence further comprises:(iii) a deletion of an endogenous non-human IgD constant region gene;(iv) a deletion of an endogenous non-human IgG3 constant region gene;(v) a deletion of an endogenous non-human IgG2a constant region gene;(vi) a deletion of an endogenous non-human IgG2b constant region gene;(vii) a deletion of an endogenous non-human IgE constant region gene;and (viii) a deletion of an endogenous non-human IgA constant regiongene.
 10. A non-human animal B cell isolated from the non-human animalof claim 1, comprising a rearranged human Ig heavy chain variable regiongene operably linked to the endogenous non-human IgG1 constant regiongene modified to (1) comprise a nucleotide sequence comprising from 5′to 3′: an endogenous non-human switch region, an IgG1 CH2 exon, an IgG1CH2 intron, and an IgG1 CH3 exon, and a deletion in a sequence encodingan IgG1 CH1 domain and an IgG1 hinge region such that the nucleotidesequence further comprises, at the deletion point, a nucleotide sequenceset forth as SEQ ID NO:17, and (2) encode an endogenous non-human IgG1constant domain that is devoid of a CH1 domain and hinge region, andthat consists essentially of an endogenous non-human IgG1 CH2 domain andan endogenous non-human IgG1 CH3 domain, wherein the rearranged human Igheavy chain variable region gene encodes a human heavy chain variabledomain, and wherein the non-human animal B cell is a rat B cell or amouse B cell.
 11. The non-human animal B cell of claim 10, wherein the Bcell secretes an IgG1 antibody comprising a chimeric human/non-humanIgG1 immunoglobulin heavy chain comprising, from N-terminal toC-terminal, a human Ig heavy chain variable domain operably linked to anendogenous non-human IgG1 constant domain that is devoid of a CH1 domainand a hinge region, and that consists essentially of an endogenousnon-human IgG1 CH2 domain and an endogenous non-human IgG1 CH3 domain.12. A human heavy chain variable domain isolated from the non-humananimal B cell of claim 10, or from a hybridoma produced therefrom.
 13. Ahybridoma made from the B cell of claim
 10. 14. A nucleic acidcomprising a rearranged human heavy chain variable region gene thatencodes a human heavy chain variable domain isolated from the non-humanB cell of claim 10, or from a hybridoma produced therefrom.
 15. A methodfor isolating a human Ig heavy chain variable domain and/or a rearrangedhuman Ig heavy chain variable region gene encoding the same comprising:obtaining a lymphocyte from the non-human animal of claim 1, orhybridoma produced therefrom, wherein the lymphocyte or hybridomaexpresses: (i) a chimeric human/non-human IgG1 immunoglobulin heavychain that comprises, from N-terminal to C-terminal, the human Ig heavychain variable domain operably linked to the endogenous non-human IgG1constant domain that is devoid of a CH1 domain and a hinge region, andthat consists essentially of an endogenous non-human IgG1 CH2 domain andan endogenous non-human IgG1 CH3 domain, or (ii) a chimerichuman/non-human IgM immunoglobulin heavy chain that comprises the humanIg heavy chain variable domain operably linked to the endogenousnon-human IgM constant domain comprising an IgM CH1 domain; andisolating the human Ig heavy chain variable domain and/or the rearrangedhuman Ig heavy chain variable region gene encoding the same from theisolated lymphocyte, or hybridoma produced therefrom.
 16. The method ofclaim 15, further comprising expressing the rearranged human Ig heavychain variable region gene encoding the human Ig heavy chain variabledomain in a host cell.
 17. A non-human animal embryonic stem (ES) cellcomprising in its genome a modified endogenous non-human Ig heavy chainlocus that comprises unrearranged human Ig heavy chain variable regionV_(H), D_(H), and J_(H) gene segments operably linked to a modifiedendogenous non-human Ig heavy chain constant region nucleic acidsequence comprising (i) an endogenous non-human IgG1 constant regiongene modified to (1) comprise a nucleotide sequence comprising from 5′to 3′: an endogenous non-human switch region, an IgG1 CH2 exon, an IgG1CH2 intron, and an IgG1 CH3 exon, and a deletion in a sequence encodingan IgG1 CH1 domain and an IgG1 hinge region such that the nucleotidesequence further comprises, at the deletion point, a nucleotide sequenceset forth as SEQ ID NO:17, and (2) encode an endogenous non-human IgG1constant domain that is devoid of a CH1 domain and hinge region, andthat consists essentially of an endogenous non-human IgG1 CH2 domain andan endogenous non-human IgG1 CH3 domain; and (ii) an endogenousnon-human IgM constant region gene that encodes an endogenous non-humanIgM constant domain comprising an IgM CH1 domain, wherein theunrearranged human Ig heavy chain variable region V_(H), D_(H), andJ_(H) gene segments are capable of recombining in a B cell of anon-human animal to form a rearranged human Ig heavy chain variableregion gene operably linked to the modified endogenous non-human Igheavy chain constant region such that the modified endogenous non-humanIg heavy chain locus in the B cell encodes i) a chimeric human/non-humanIgG1 immunoglobulin heavy chain that comprises, from N-terminal toC-terminal, a human Ig heavy chain variable domain operably linked tothe endogenous non-human IgG1 constant domain that is devoid of a CH1domain and a hinge region, and that consists essentially of anendogenous non-human IgG1 CH2 domain and an endogenous non-human IgG1CH3 domain, and (ii) a chimeric human/non-human IgM immunoglobulin heavychain that comprises a human Ig heavy chain variable domain operablylinked to the endogenous non-human IgM constant domain comprising an IgMCH1 domain, and wherein the non-human animal ES cell is a rat ES cell ora mouse ES cell, and the non-human animal is a rat or a mouse.
 18. Thenon-human animal ES cell of claim 17, wherein the ES cell is from amouse strain selected from the group consisting of a 129 strain, aC57BL/6 strain, and a mixed 129xC57BL/6 strain.
 19. The non-human animalES cell of claim 18, wherein the ES cell is 50% 129 and 50% C57BL/6. 20.A mouse embryo made from or comprising the non-human animal ES cell ofclaim
 18. 21. A non-human animal embryo made from or comprising thenon-human animal ES cell of claim 17, wherein the non-human animalembryo is a rat embryo or a mouse embryo.
 22. The non-human animal EScell of claim 17, wherein the modified endogenous non-human Ig heavychain constant region nucleic acid sequence further comprises a deletionof an endogenous non-human IgG2a constant region gene and a deletion ofan endogenous non-human IgG2b constant region gene.
 23. The non-humananimal ES cell of claim 17, wherein the modified endogenous non-human Igheavy chain constant region nucleic acid sequence further comprises:(iii) a deletion of an endogenous non-human IgD constant region gene;(iv) a deletion of an endogenous non-human IgG3 constant region gene;(v) a deletion of an endogenous non-human IgG2b constant region gene;(vi) a deletion of an endogenous non-human IgE constant region gene; and(vii) a deletion of an endogenous non-human IgA constant region gene.24. The non-human animal ES cell of claim 17, wherein the modifiedendogenous non-human Ig heavy chain constant region nucleic acidsequence further comprises: (iii) a deletion of an endogenous non-humanIgD constant region gene; (iv) a deletion of an endogenous non-humanIgG3 constant region gene; (v) a deletion of an endogenous non-humanIgG2a constant region gene; (vi) a deletion of an endogenous non-humanIgG2b constant region gene; (vii) a deletion of an endogenous non-humanIgE constant region gene; and (viii) a deletion of an endogenousnon-human IgA constant region gene.